Novel polyketide derivatives and recombinant methods for making same

Abstract

The invention provides novel erythromycin derivatives in which methyl groups on the macrolactone ring have been substituted with —H, -Et, and/or —OH. The invention also provides reagents such as isolated polynucleotides, vectors comprising the polynucleotides and host cells transformed with the vectors for making the novel compounds. Methods for making the compounds utilizing genetic engineering techniques are also disclosed.

What is claimed is:  
     
         1 . A compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 R 1 , R 2,  R 3,  R 4,  R 5,  and R 6  are independently selected from Q wherein Q is selected from the group consisting of (a) —H, (b) -Me, (c) -Et, and (d) —OH;  
 L 1  and L 2  are independently —H or —OH;  
 L 3  is D-desosamine or —OH; and  
 L 4  is L-mycarose, L-cladinose or —OH with the proviso that when R 1 -R 5  are -Me, R 6  is other than —H or -Me.  
 
     
     
         2 . The compound of  claim 1  wherein Q is selected from the group consisting of (a), (b), and (c) and L 1 , L 2,  L 3  and L 4  are as defined therein.  
     
     
         3 . The compound of  claim 1  wherein Q is selected from the group consisting of (a), (b), and (d) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         4 . The compound of  claim 1  wherein Q is selected from the group consisting of (a), (c), and (d) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         5 . The compound of  claim 1  wherein Q is selected from the group consisting of (b), (c), and (d) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         6 . The compound of  claim 1  wherein Q is selected from the group consisting of (a) and (b) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         7 . The compound of  claim 1  wherein Q is selected from the group consisting of (a) and (c) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         8 . The compound of  claim 1  wherein Q is selected from the group consisting of (a) and (d) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         9 . The compound of  claim 1  wherein Q is selected from the group consisting of (b) and (c) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         10 . The compound of  claim 1  wherein Q is selected from the group consisting of (b) and (d) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         11 . The compound of  claim 1  wherein Q is selected from the group consisting of (c) and (d) and L 1,  L 2,  L 3  and L 4  are as defined therein.  
     
     
         12 . The compound of  claim 1  wherein Q is (a) and L 1 , L 2,  L 3  and L 4  are is defined therein.  
     
     
         13 . The compound of  claim 1  wherein Q is (c) and L 1 , L 2,  L 3  and L 4  are as defined therein.  
     
     
         14 . The compound of  claim 1  wherein Q is (d) and L 1 , L 2,  L 3  and L 4  are as defined therein.  
     
     
         15 . The compound of  claim 1  wherein 
 (a) R 6  and R 1  are —H and R 2 , R 3 , R 4  and R 5  are -Me,  
 (b) R 5  and R 1  are —H and R 2 , R 3 , R 4  and R 6  are -Me,  
 (c) R 4  and R 1  are —H and R 2 , R 3 , R 5  and R 6  are -Me,  
 (d) R 3  and R 1  are —H and R 2 , R 4 , R 5  and R 6  are -Me,  
 (e) R 2  and R 1  are —H and R 3 , R 4 , R 5  and R 6  are -Me,  
 (f) R 6  and R 2  are —H and R 1 , R 3 l , R   4  and R 5  are -Me,  
 (g) R 5  and R 2  are —H and R 1 , R 3 , R 4  and R 6  are -Me,  
 (h) R 4  and R 2  are —H and R 1 , R 3 , R 5  and R 6  are -Me,  
 (i) R 3  and R 2  are —H and R 1 , R 4 , R 5  and R 6  are -Me,  
 (j) R 6  and R 3  are —H and R 1 , R 2 , R 4  and R 5  are -Me,  
 (k) R 5  and R 3  are —H and R 1 , R 2 , R 4  and R 6  are -Me,  
 (l) R 4  and R 3  are —H and R 1 , R 2 , R 5  and R 6  are -Me,  
 (m) R 6  and R 4  are —H and R  1 , R 2 , R 3  and R 5  are -Me,  
 (n) R 5  and R 4  are —H and R  1 , R 2 , R 3  and R 6  are -Me,  
 (o) R 6  and R 5  are —H and R  2 , R 3  and R 4  are -Me, and L 1 , L 2 , L 3  and L 4  are as defined therein.  
 
     
     
         16 . The compound of  claim 15  wherein (a)-(o) are as defined therein, L 1  and L 2  are —OH, L 3  is D-desosamine and L 4  is L-cladinose.  
     
     
         17 . The compound of  claim 1  wherein 
 (a) R 6 , R 2  and R 1  are —H and R 3 , R 4  and R 5  are -Me,  
 (b) R 5 , R 2  and R 1  are —H and R 3 , R 4  and R 6  are -Me,  
 (c) R 4 , R 2  and R 1  are —H and R 3 , R 5  and R 6  are -Me,  
 (d) R 3 , R 2  and R 1  are —H and R 4 , R 5  and R 6  are -Me,  
 (e) R 6 , R 3  and R 1  are —H and R 2 , R 4  and R 5  are -Me,  
 (f) R 5 , R 3  and R 1  are —H and R 2 , R 4  and R 6  are -Me,  
 (g) R 4 , R 3  and R 1  are —H and R 2 , R 5  and R 6  are -Me,  
 (h) R 6 , R 4  and R 1  are —H and R 2 , R 3  and R 5  are -Me,  
 (i) R 5 , R 4  and R 1  are —H and R 2 , R 3  and R 6  are -Me,  
 (j) R 6 , R 5  and R 1  are —H and R 2 , R 3  and R 4  are -Me,  
 (k) R 6 , R 3  and R 2  are —H and R 1 , R 4  and R 5  are -Me,  
 (l) R 5 , R 3  and R 2  are —H and R 1 , R 4  and R 6  are -Me,  
 (m) R 4 , R 3  and R 2  are —H and R 1 , R 5  and R 6  are -Me,  
 (n) R 6 , R 4  and R 2  are —H and R 1 , R 3  and R 5  are -Me,  
 (o) R 5 , R 4  and R 2  are —H and R 1 , R 3  and R 6  are -Me,  
 (p) R 6 , R 5  and R 2  are —H and R 1 , R 3  and R 6  are -Me,  
 (q) R 6 , R 4  and R 3  are —H and R 1 , R 2  and R 5  are -Me,  
 (r) R 5 , R 4  and R 3  are —H and R 1 , R 2  and R 6  are -Me,  
 (s) R 6 , R 5  and R 3  are —H and R 1 , R 2  and R 4  are -Me, or  
 (t) R 6 , R 5  and R 4  are —H and R 1 , R 2  and R 3  are -Me, and L 1 , L 2 , L 3  and L 4  are as defined therein.  
 
     
     
         18 . The conmpound of  claim 17  wherein (a)-(t) are as defined therein, L 1  and L 2  are —OH, L 3  is D-desosamine and L 4  is L-cladinose.  
     
     
         19 . The compound of  claim 1  wherein 
 (a) R 6 , R 3 , R 2  and R 1  are —H and R 5 , and R 4  are -Me,  
 (b) R 5 , R 3 , R 2  and R 1  are —H and R 6 , and R 4  are -Me,  
 (c) R 4 , R 3 , R 2  and R 1  are —H and R 5 , and R 6  are -Me,  
 (d) R 6 , R 4 , R 2  and R 1  are —H and R 3 , and R 5  are -Me,  
 (e) R 5 , R 4 , R 2  and R 1  are —H and R 3 , and R 6  are -Me,  
 (f) R 6 , R 5 . R 2  and R 1  are —H and R 3 , and R 4  are -Me,  
 (g) R 6 , R 4 , R 3  and R 1  are —H and R 2 , and R 5  are -Me,  
 (h) R 5 , R 4 , R 3  and R 1  are —H and R 2 , and R 6  are -Me,  
 (i) R 6 , R 5 , R 4  and R 1  are —H and R 2 , and R 3  are -Me,  
 (j) R 2 , R 4 , R 3  and R 2  are —H and R 1 , and R 6  are -Me,  
 (k) R 6 , R 4 , R 3  and R 2  are —H and R 1 , and R 6  are -Me,  
 (l) R 6 , R 4 , R 3  and R 2  are —H and R 1 , and R 6  are -Me,  
 (m) R 6 , R 5 , R 3  and R 2  are —H and R 1 , and R 4  are -Me, or  
 (n) R 6 , R 5 , R 4  and R 3  are —H and R 1 , and R 2  are -Me, and L 1 , L 2 , L 3  and L 4  are as defined therein.  
 
     
     
         20 . The compound of claim 19 wherein (a)-(n) are as defined therein, L 1  and L 2  are —OH, L 3  is D-desosamine and L 4  is L-cladinose.  
     
     
         21 . The compound of  claim 1  wherein 
 (a) R 5 , R 4 , R 3 , R 2  and R 1  are —H and R 6  is -Me,  
 (b) R 6 , R 4 , R 3 , R 2  and R 1  are —H and R 5  is -Me,  
 (c) R 6 , R 5 , R 3 , R 2  and R 1  are —H and R 4  is -Me,  
 (d) R 6 , R 5 , R 4 , R 2  and R 1  are —H and R 3  is -Me,  
 (e) R 6 , R 5 , R 4 , R 3  and R 2  are —H and R 2  is -Me, or  
 (f) R 6 , R 5 , R 4 , R 3  and R 2  are —H and R 1  is -Me, and L 1 , L 2 , L 3  and L 4  are as defined therein.  
 
     
     
         22 . The compound of  claim 21  wherein (a)-(f) are as defined therein, L 1  and L 2  are —OH, L 3  is D-desosamine and L 4  is L-cladinose.  
     
     
         23 . The compound of  claim 1  wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6  are —H and L 1 , L 2 , L 3  and L 4  are as defined therein.  
     
     
         24 . The compound of  claim 23  wherein R 1,  R 2,  R 3 , R 4 , R 5  and R 6  are as defined therein, L 1  and L 2  are —OH, L 3  is D-desosamine and L 4  is L-cladinose.  
     
     
         25 . The compound of  claim 1  selected from the group consisting of 6,10-didesmethlyl-6-ethylerythromycin A; 10,12-didesmethyl-12-deoxy-12-ethylerythromycin A; 10,12-didesmethyl-12-deoxy-10-hydroxyerythromycin A; 6,10,12-tridesmethyl-6,12-diethylerythromycin A, and 6,10,12-tridesmethyl-6-deoxy-6,12-diethylerythlromycin A.  
     
     
         26 . The compound of  claim 1  selected from the group consisting of 10-desmethylerythronolide B, 10-desmethyl-6-deoxyerythronolide B, 12-desmethylerythronolide B, 12-desmethyl-6-deoxyerythronolide B, 12-desmethyl-12-ethylerythronolide B, 6-desmethyl-6-deoxy-6-ethylerythronolide B, 10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A, 10-desmethyl-6,12-dideoxyerythromycin A, 12-desmethylerythromycin A, 12-desmethyl- 12-deoxyerythromycin A, 12-desmethyl-6,12-dideoxyerythromycin A, 6-desmethyl-6-6-ethtlerthromycin A, 12-desmethyl-12-ethylerythromycin A, 12-desmethyl-12-deoxy-12-ethylerythromycin A, 10-desmethyl-10-hydroxyerythromycin A, 12-desmethyl-12-epihydroxyerythromycin A, 10,12-didesmethylerythromycin A, 10,12-didesmethyl-12-deoxyerythromycin A, and 10,12-didesmethyl-6,12-dideoxyerythromycin A.  
     
     
         27 . The compound of  claim 1  selected from the group consisting of 10-desmethylerthronolide B, 10-desmethyl-6-deoxyerythronolide B, 12-desmethylerythronolide B, 12-desmethyl-6-deoxyerythronolide B 10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A, 10-desmethyl-6,12-deoxyerythromycin A, 12-desmethyl-6,12-dideoxyerythromycin A, 10,12-didesmethylerythromycin A, 10,12didesmethyl-12-deoxyerythromycin A, 10,12-didesmethyl-6,12-didteoxyerythromycin A.  
     
     
         28 . A compound selected from the group consisting of 10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A, and 12-desmethyl-12-deoxyerythromycin A.  
     
     
         29 . An isolated polynucleotide sequence or fragment thereof which encodes an enzymatically active acyltransferase domain from a polyketide-producing microorganism selected from the group consisting of  Streptomyces hygroscopicus, Streptomyces venezuelae , and  Streptomyces caelestis.    
     
     
         30 . The polynucleotide of  claim 29  selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:29 and SEQ ID NO:30.  
     
     
         31 . The polynucleotide of  claim 29  wherein said acyltransferase domain is selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.  
     
     
         32 . A vector comprising a polynucleotide sequence or fragment thereof which encodes an enzymatically active acyltransferase domain from Streptomyces.  
     
     
         33 . The vector of  claim 32  wherein said Streptomyces is selected from the group consisting of  Streptomyces hygroscopicus, Streptomyces venezuelae , and  Streptomtces caelestis.    
     
     
         34 . The vector of  claim 32  which is pCS5.  
     
     
         35 . The vector of  claim 32  wherein said polynucleotide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:29 and SEQ ID NO:30.  
     
     
         36 . The vector of  claim 32  wherein said acyltransferase domain is selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.  
     
     
         37 . A vector selected from the group consisting of pUC18/LigAT2, pEryAT1/LigAT2, pEryAT2/LigAT2, pUC18/venAT, pEryAT1/venAT, pUC19/rapAT14, pEryAT1/rapAT14, pEryAT2/rapAT14, pUC/5′-flank/ethAT, pUC/ethAT/C-6, pEAT4, pUC18/NidAT6, and pEryAT2/NidAT6.  
     
     
         38 . A host cell transformed with the vector of  claim 32 .  
     
     
         39 . The host cell of  claim 38  wherein said cell is a bacterial cell.  
     
     
         40 . The host cell of  claim 39  wherein said bacterial cell is selected from the group consisting of  E. coli  and BacilIus species.  
     
     
         41 . The host cell of  Claim 40  wherein said cell is a polyketide-producing microorganism.  
     
     
         42 . The host cell of  claim 41  wherein said polyketide-producing microorganism is selected from the group consisting of Saccharopolyspora species, Nocardia species, Micromonospor species, Arthrobacter species, Streptomyces species, Actinomadura species, and Dactylosporangium species.  
     
     
         43 . The host cell of  claim 42  wherein said polyketide-producing microorgianism is selected from the group consisting of  Saccharopolyspora hirsuta, Micromonospora rosaria, Micromonospora megalomicea, Streptomyces antibioticus, Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyces hygroscopicus, Streptomyces caelestis, Streptomyces tsukubaensis, Streptomyces fradiae, Streptomyces plantensis, Streptomyces violaceoniger, Streptomyces ambofaciens, Streptomyces griseoplanus , and  Streptomyces venezuelae.    
     
     
         44 . The host cell of  claim 42  wherein said polyketide-producing microorganism is selected from the group consisting of Saccharopolyspora species and Streptomyces species.  
     
     
         45 . The host cell of  claim 44  wherein said polyketide-producing microorganism is  Saccharopolyspora erythraea.    
     
     
         46 . The host cell of  claim 44  wherein said polyketide-producing microorganism is selected from the group consisting of  Streptomyces hygroscopicus, Streptomyces venezuelae , and  Streptomyces caelestis.    
     
     
         47 . A method for altering the substrate specificity of a polykeitde synthase in a first polyketide-producing microorganism comprising the steps of : 
 (a) isolating a first and second genomic DNA segment, each comprising a polyketide synthase wherein said first geomic DNA segment is from said first polyketide-producing microorganism and said second genomic DNA segment is from said first polykeitde-producing microorganism or a second polyketide-producing microorganism;    (b) identifying one of more descrete fragments of said first genomic DNA segment, each of which encodes an acyltransferase domain:    (c) identifying one or more discrete fragments of said second genemic DNA segment, each of which encodes a related domain to said acyltranseferase domain of said first genomic DNA segment; and    (d) transforming a cell of said first polyketide-producing microorganism with one or more of said fragments from step (c) under conditions suitable for the occurrence of a homologous recombination event, leading to the replacement of one or more of said fragments from said first genomic DNA segment with one or more of said fragments from step (c).    
     
     
         48 . The method of  claim 47  wherein said first polyketide-producing microorganism is  Saccharopolyspora erythraea.    
     
     
         49 . The method of  claim 47  wherein said second polyketide-producing microorganism is Streptomyces.  
     
     
         50 . The method of  claim 49  wherein said Streptomyces is selected from the group consisting of  Streptomyces antibioticus, Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyces hygroscopicus, Streptomyces caelestis, Streptomyces tsukubaensis, Streptomyces fradiae, Streptomyces platensis, Streptomyces violaceoniger, Streptomyces ambofaciens , and  Streptomyces venezuelae.    
     
     
         51 . The method of  claim 50  wherein said Streptomyces is selected from the group consisting of  Streptomyces caelestis, Streptomyces hygroscopicus , and  Streptomyces venezuelae.    
     
     
         52 . The method of  claim 47  wherein said first polyketide-producing microorganism is Streptomyces.  
     
     
         53 . The method of  claim 52  wherein said Streptomyces is selected from the group consisting of  Streptomyces antibioticus, Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyces hygroscopicus, Streptomyces caelestis, Streptomyces tsukubaensis, Streptomyces fradiae, Streptomyces platensis, Streptomyces violaceoniger, Streptomyces ambofaciens , and  Streptomyces venezuelae.    
     
     
         54 . The method of  claim 53  wherein said Streptomyces is selected from the group consisting of  Streptomyces caelestis, Streptomyces hygroscopicus , and  Streptomyces venezuelae.    
     
     
         55 . The method of  claim 47  wherein said second polyketide-producing microorganism is  Saccharopolyspora erythraea.    
     
     
         56 . The method of  claim 47  wherein said related domain is selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.