Overproduction hosts for biosynthesis of polyketides

Abstract

Generic overproduction host cells can be used to produce any polyketide and obviate the need for performing conventional strain improvement.

1 . An overproducing host cell comprising polyketide synthase (PKS) genes encoding a first PKS that produce a first polyketide, wherein the cell has been optimized for production of a second polyketide, wherein the cell produces the second polyketide at a level greater than 1 g/L of culture medium, and wherein the genes encoding the second PKS are deleted or otherwise rendered inactive.  
     
     
         2 . The overproducing host cell of  claim 1 , wherein the genes encoding the second PKS are deleted or otherwise rendered inactive prior to introduction of the genes that encode the PKS that produce the first polyketide.  
     
     
         3 . The overproducing host cell of  claim 1 , wherein the genes encoding the second PKS are deleted or otherwise rendered inactive after the introduction of the genes that encode the PKS that produce the first polyketide.  
     
     
         4 . The overproducing host cell of  claim 1 , wherein the genes encoding the second PKS are deleted or otherwise rendered inactive during the introduction of the genes that encode the PKS that produce the first polyketide.  
     
     
         5 . An overproducing host cell comprising polyketide synthase (PKS) genes encoding a first PKS that produces a first polyketide, wherein the cell has been optimized for production of a second polyketide, wherein the cell produces the second polyketide at a level greater than 10 g/L of culture medium, and wherein the genes encoding the second PKS are deleted or otherwise rendered inactive.  
     
     
         6 . The overproducing host cell of  claim 5 , wherein the genes encoding the second PKS are deleted or otherwise rendered inactive prior to introduction of the genes that encode the PKS that produce the first polyketide.  
     
     
         7 . The overproducing host cell of  claim 5 , wherein the genes encoding the second PKS are deleted or otherwise rendered inactive after the introduction of the genes that encode the PKS that produce the first polyketide.  
     
     
         8 . The overproducing host cell of  claim 5 , wherein the genes encoding the second PKS are deleted or otherwise rendered inactive during the introduction of the genes that encode the PKS that produce the first polyketide.  
     
     
         9 . The overproducing host cell of  claim 1  or  5 , wherein the first polyketide is 10,11-anhydro-6-deoxyerythronolide B.  
     
     
         10 . The overproducing host cell of  claim 1  or  5 , wherein the first polyketide is a derivative of the second polyketide.  
     
     
         11 . A modified overproducing host cell comprising polyketide synthase (PKS) genes encoding a first PKS that produce a first polyketide, wherein the cell has been optimized for the production of erythromycin, and wherein the cell is derived from a  Saccharopolyspora erythraea  host cell that produces erythromycin at a level greater than 2.5 g/L of culture medium.  
     
     
         12 . The overproducing host cell of  claim 11 , wherein the modification is the deletion of all or substantially all of the eryA genes.  
     
     
         13 . The overproducing host cell of  claim 11 , wherein the modification is the inactivation of the ketosynthase (KS) domain of module 1 of the 6-deoxyerythronolide B synthase (DEBS) PKS.  
     
     
         14 . A recombinant host cell wherein the DNA present in the recombinant host cell has been modified, relative to the DNA of the cell from which it was derived, to contain one or more attB or attP sites.  
     
     
         15 . The recombinant host cell of  claim 14  that is derived from a cell that has been optimized by mutagenesis to produce a polyketide at a level higher than before optimization.  
     
     
         16 . The recombinant host cell of  claim 14  that is a  Saccharopolyspora erythraea  host cell.  
     
     
         17 . The recombinant host cell of  claim 16  that is derived from  Saccharopolyspora erythraea  NRRL2338.  
     
     
         18 . The recombinant host cell of  claim 16  that is derived from a cell that produces erythromycins at a level greater than 2.5 g/L of culture medium.  
     
     
         19 . A method for obtaining a transformant of a recombinant host cell of  claim 14 , which comprises conjugating said recombinant host cell with another cell that contains a vector that comprises an attachment site complementary to attB or attP, in the presence of an integrase.  
     
     
         20 . A transformant produced by the method of  claim 19 .  
     
     
         21 . The transformant of  claim 20  that produces a polyketide that is not produced by either of the cells prior to said conjugating step.