Genes from a gene cluster

Abstract

ML-236B is an inhibitor of HMG-CoA reductase and useful in preparing another such inhibitor, pravastatin. The preparation of ML-236B using an ML-236B-producing microorganism is enhanced using a polynucleotide encoding a gene related to the polyketide synthase cluster occurring in such a microorganism.

1 . A polynucleotide selected from the group consisting of: 
 (a) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 38, or a polynucleotide variant thereof encoding a modified amino acid sequence having at least one deletion, addition, substitution or alteration, said polynucleotide variant being capable of accelerating the biosynthesis of ML-236B; and    (b) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 42, or a polynucleotide variant thereof encoding a modified amino acid sequence having at least one deletion, substitution or alteration, said polynucleotide variant being capable of accelerating the biosynthesis of ML-236B.    
     
     
         2 . A polynucleotide according to  claim 1  comprising SEQ ID NO: 37 or a mutant or variant of SEQ ID NO: 37 capable of accelerating the biosynthesis of ML-236B.  
     
     
         3 . A polynucleotide according to  claim 1  comprising SEQ ID NO: 37.  
     
     
         4 . A polynucleotide according to  claim 1  comprising DNA obtained from transformed  Escherichia coli  pSAKexpE SANK 72499 (FERM BP-7005).  
     
     
         5 . A polynucleotide according to  claim 1  comprising SEQ ID NO: 41 or a variant of SEQ ID NO: 41 capable of accelerating the biosynthesis of ML-236B.  
     
     
         6 . A polynucleotide according to  claim 1  comprising SEQ ID NO: 41.  
     
     
         7 . A polynucleotide according to  claim 1  comprising DNA obtained from transformed  Escherichia coli  pSAKexpR SANK 72599 (FERM BP-7006).  
     
     
         8 . A polynucleotide according to  claim 1  in combination with one or more other polynucleotides, said combination being capable of enhancing the production of ML-236B in a ML-236B producing micro-organism.  
     
     
         9 . A polynucleotide according to  claim 3  in combination with one or more other polynucleotides, said combination being capable of enhancing the production of ML-236B in a ML-236B producing micro-organism.  
     
     
         10 . A polynucleotide according to  claim 4  in combination with one or more other polynucleotides, said combination being capable of enhancing the production of ML-236B in a ML-236B producing micro-organism.  
     
     
         11 . A polynucleotide according to  claim 6  in combination with one or more other polynucleotides, said combination being capable of enhancing the production of ML-236B in a ML-236B producing micro-organism.  
     
     
         12 . A polynucleotide according to  claim 7  in combination with one or more other polynucleotides, said combination being capable of enhancing the production of ML-236B in a ML-236B producing micro-organism.  
     
     
         13 . A polynucleotide according to  claim 8  comprising a polynucleotide of SEQ ID NO: 37, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         14 . A polynucleotide according to  claim 9  comprising a polynucleotide of SEQ ID NO: 37, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         15 . A polynucleotide according to  claim 10  comprising a polynucleotide of SEQ ID NO: 37, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         16 . A polynucleotide according to  claim 11  comprising a polynucleotide of SEQ ID NO: 37, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID: NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         17 . A polynucleotide according to  claim 12  comprising a polynucleotide of SEQ ID NO: 37, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         18 . A polynucleotide according to  claim 8  comprising a polynucleotide of SEQ ID NO: 41, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         19 . A polynucleotide according to  claim 9  comprising a polynucleotide of SEQ ID NO: 41, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         20 . A polynucleotide according to  claim 10  comprising a polynucleotide of SEQ ID NO: 41, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         21 . A polynucleotide according to  claim 11  comprising a polynucleotide of SEQ ID NO: 41, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         22 . A polynucleotide according to  claim 12  comprising a polynucleotide of SEQ ID NO: 41, or a variant thereof, in combination with one or more sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and variants thereof.  
     
     
         23 . A polynucleotide capable of hybridizing under stringent conditions with a polynucleotide according to  claim 1 .  
     
     
         24 . A polynucleotide according to  claim 23  capable of accelerating the biosynthesis of ML-236B in a ML-236B producing micro-organism when introduced in the ML-236B producing micro-organism.  
     
     
         25 . A polynucleotide according to  claim 23  which is RNA.  
     
     
         26 . A vector comprising a polynucleotide according to  claim 1 .  
     
     
         27 . A vector according to  claim 26  obtained from  Escherichia coli  pSAKexpE SANK 72499 (FERM BP-7005) or  Escherichia coli  pSAKexpR SANK 72599 (FERM BP-7006).  
     
     
         28 . A vector according to  claim 26  which is an expression vector.  
     
     
         29 . A host cell transformed by a vector according to  claim 26 .  
     
     
         30 . A host cell according to  claim 29 , wherein the host cell is a ML-236B producing micro-organism.  
     
     
         31 . A host cell according to  claim 30 , wherein the host cell is  Penicillium citrinum.    
     
     
         32 . A host cell according to  claim 29 , wherein the host cell is  Escherichia coli.    
     
     
         33 . A host cell according to  claim 32 , wherein the host cell is  Escherichia coli  pSAKexpE SANK 72499 (FERM BP-7005).  
     
     
         34 . A host cell according to  claim 32 , wherein the host cell is  Escherichia coli  pSAKexpR SANK 72599 (FERM BP-7006).  
     
     
         35 . A polypeptide encoded by a polynucleotide according to  claim 1 .  
     
     
         36 . A polypeptide comprising the sequence of SEQ ID NO: 38, or a variant thereof which has at least 80% identity to SEQ ID NO: 38 and which is capable of accelerating ML-236B production in an ML-236B producing organism.  
     
     
         37 . A polypeptide according to  claim 36 , having the sequence of SEQ ID NO: 38.  
     
     
         38 . A polypeptide comprising the sequence of SEQ ID NO: 42, or a variant thereof which has at least 80% identity with SEQ ID NO: 42 and which is capable of accelerating ML-236B production in an ML-236B producing organism.  
     
     
         39 . A polypeptide according to  claim 38 , having the sequence of SEQ ID NO: 42.  
     
     
         40 . A method for producing ML-236B comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by a vector comprising a polynucleotide sequence encoding mlcE, wherein said vector does not comprise a polynucleotide sequence encoding at least one of mlcA, mlcB, mlcC, and mlcD and    (b) recovering ML-236B from the resultant culture; wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium;      and wherein mlcE has the amino acid sequence of SEQ ID NO: 38, mlcA has the amino acid sequence of SEQ ID NO: 44, mlcB has the amino acid sequence of SEQ ID NO: 46, mlcC has the amino acid sequence of SEQ ID NO: 48, and mlcD has the amino acid sequence of SEQ ID NO: 50.    
     
     
         41 . The method according to  claim 40 , wherein the host cell is transformed with a vector comprising a polynucleotide having the nucleotide sequence SEQ ID NO: 37.  
     
     
         42 . A method of manufacturing pravastatin which comprises carrying out the method according to  claim 40  and converting the ML-236B to pravastatin.  
     
     
         43 . The method according to  claim 40 , wherein the polynucleotide encodes a protein consisting of the amino acid sequence of SEQ ID NO: 38.  
     
     
         44 . The method according to  claim 40 , wherein the polynucleotide is a cDNA.  
     
     
         45 . The method according to  claim 40 , wherein the polynucleotide is a genomic DNA.  
     
     
         46 . A method for producing ML-236B comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by a vector comprising a polynucleotide sequence encoding mlcE, wherein said vector does not comprise a polynucleotide sequence encoding at least one of mlcA, mlcB, mlcC, and mlcD and    (b) recovering ML-236B from the resultant culture;    wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium;      wherein mlcE has the amino acid sequence of SEQ ID NO: 38;    and wherein said vector does not comprise at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49.    
     
     
         47 . A method or producing ML-236B comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by a vector comprising a polynucleotide sequence encoding mlcE, and    (b) recovering ML-236B from the resultant culture;    wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium;      and wherein mlcE has the amino acid sequence of SEQ ID NO: 38, and wherein said producing occurs in the absence of a recombinant polynucleotide sequence encoding at least one of mlcA, mlcB, mlcC, and mlcD and wherein mlcA has the amino acid sequence of SEQ ID NO: 44, mlcB has the amino acid sequence of SEQ ID NO: 46, mlcC has the amino acid sequence of SEQ ID NO: 48, and mlcD has the amino acid sequence of SEQ ID NO: 50.    
     
     
         48 . A method for producing ML-236B comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by pSAKexpE and    (b) recovering ML-236B from the resultant culture;    wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium.      
     
     
         49 . A method of manufacturing pravastatin comprising carrying out the method according to  claim 48  and converting the ML-236B to pravastatin.  
     
     
         50 . A method for producing ML-236B comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by a vector comprising a polynucleotide sequence encoding mlcE, and    (b) recovering ML-236B from the resultant culture;    wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium;      and wherein mlcE has the amino acid sequence of SEQ ID NO: 38, and wherein said producing occurs in the absence of at least one nucleotide sequence from the group consisting of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49.    
     
     
         51 . An antibody reactive with the protein of SEQ ID NO: 38 or SEQ ID NO: 42.  
     
     
         52 . A polynucleotide encoding protein having the amino acid sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50 or a variant polynucleotide encoding a modification of said amino acid sequence having a deletion, substitution, addition or alteration, said variant being suitable for use in accelerating the biosynthesis of ML-236B.  
     
     
         53 . A polynucleotide according to  claim 52 , wherein the polynucleotide is selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49.  
     
     
         54 . A polynucleotide according to  claim 52 , wherein said polynucleotide is capable of accelerating the biosynthesis of ML-236B alone or in conjunction with the polynucleotide of SEQ ID NO: 37 or SEQ ID NO: 41.  
     
     
         55 . A vector comprising a polynucleotide according to  claim 52 .  
     
     
         56 . A host cell comprising a vector according to  claim 55 .  
     
     
         57 . A polypeptide encoded by a polypeptide according to  claim 52 .  
     
     
         58 . A method for the production of ML-236B comprising culturing a host cell according to  claim 56  and then recovering ML-236B from the culture.  
     
     
         59 . ML-236B produced by a method comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by a vector comprising a polynucleotide sequence encoding mlcR or mlcE, wherein said vector does not comprise a polynucleotide sequence encoding at least one of mlcA, mlcB, mlcC, and mlcD and    (b) recovering ML-236B from the resultant culture;    wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium;      and wherein mlcR has the amino acid sequence of SEQ ID NO: 42, wherein mlcE has the amino acid sequence of SEQ ID NO: 38, mlcA has the amino acid sequence of SEQ ID NO: 44, mlcB has the amino acid sequence of SEQ ID NO: 46, mlcC has the amino acid sequence of SEQ ID NO: 48, and mlcD has the amino acid sequence of SEQ ID NO: 50.    
     
     
         60 . ML-236B produced by a method comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by a vector comprising a polynucleotide sequence encoding mlcR or mlcE, wherein said vector does not comprise a polynucleotide sequence encoding at least one of mlcA, mlcB, mlcC, and mlcD and    (b) recovering ML-236B from the resultant culture;    wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium;      wherein mlcR has the amino acid sequence of SEQ ID NO: 42 and mlcE has the amino acid sequence of SEQ ID NO: 38;    and wherein said vector does not comprise at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49.    
     
     
         61 . ML-236B produced by a method comprising: 
 (a) culturing a  Penicillium  host cell having been transformed by a vector comprising a polynucleotide sequence encoding mlcR or mlcE, and    (b) recovering ML-236B from the resultant culture;    wherein said  Penicillium  host cell is selected from the group consisting of  Penicillium citrinum, Penicillium brevicompactum  and  Penicillium cyclopium;      and wherein mlcR has the amino acid sequence of SEQ ID NO: 42 and mlcE has the amino acid sequence of SEQ ID NO: 38, and wherein said producing occurs in the absence of a recombinant polynucleotide sequence encoding at least one of mlcA, mlcB, mlcC, and mlcD and wherein mlcA has the amino acid sequence of SEQ ID NO: 44, mlcB has the amino acid sequence of SEQ ID NO:  46 , mlcC has the amino acid sequence of SEQ ID NO: 48, and mlcD has the amino acid sequence of SEQ ID NO: 50.