Modular polyketide synthases with altered activity

Abstract

The substrate specificity of modular polyketide synthases is altered by mutating or replacing acyltransferase regions thereof.

1 . A method for altering the substrate specificity of a polyketide 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 genomic DNA segment is from said first polyketide-producing microorganism and said second genomic DNA segment is from said first polyketide-producing microorganism or a second polyketide-producing microorganism;    (b) identifying one or more discrete fragments of said first genomic DNA segment, each of which encodes an acyltransferase domain;    (c) identifying one or more discrete fragments of said second genomic DNA segment, each of which encodes a related domain to said acyltransferase 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).    
     
     
         2 . A method for altering the substrate specificity of a first polyketide synthase comprising the steps of: 
 (a) isolating a first and second genomic DNA segment, each comprising a polyketide synthase wherein said first genomic DNA segment is from said first polyketide synthase and said second genomic DNA segment is from a different region of said first polyketide synthase or from a second polyketide synthase;    (b) identifying one or more discrete fragments of said first genomic DNA segment, each of which encodes an acyltransferase domain;    (c) identifying one or more discrete fragments of said second genomic DNA segment, each of which encodes an acyltransferase domain that has a different substrate specificity from that of the acyltransferase domain of said first genomic DNA segment; and    (d) transforming a cell containing said first polyketide synthase with one or more of said fragments from step (c) under conditions suitable for the occurrence of a homologous recombination event, wherein one or more of said fragments from said first genomic DNA segment is replaced by one or more of said fragments from step (c).    
     
     
         3 . A method for altering the substrate specificity of a first polyketide synthase comprising the steps of: 
 (a) identifying one or more fragments of said first polyketide synthase, each of which encodes an acyltransferase domain;    (b) identifying one or more fragments of a second polyketide synthase, each of which encodes an acyltransferase domain that has a different substrate specificity from that of the acyltransferase domain of said first polyketide synthase; and    (c) transforming a cell containing said first polyketide synthase with one or more fragments from step (b) under conditions suitable for the occurrence of a homologous recombination event, wherein one or more of said fragments from said first polyketide synthase is replaced by one or more of said fragments from step (b).    
     
     
         4 . A method for directing the biosynthesis of specific macrolide polyketide analogs by genetic manipulation of a polyketide-producing microorganism, said method comprising the steps of: 
 (1) isolating a macrolide polyketide biosynthetic gene-containing DNA sequence;    (2) identifying enzymatic activities associated within said gene-containing DNA sequence;    (3) introducing one or more specified changes into said gene-containing DNA sequence which codes for one of said enzymatic activities resulting in an altered DNA sequence;    (4) introducing said altered DNA sequence into a polyketide-producing microorganism to replace the original sequence;    (5) growing a culture of the altered microorganism under conditions suitable for the formation of the specific macrolide polyketide analog; and    (6) isolating said specific macrolide polyketide analog from the culture.    
     
     
         5 . A method to prepare a nucleotide sequence encoding a modified PKS from a nucleotide sequence encoding a naturally occurring modular PKS wherein said naturally occurring modular PKS contains first regions which encode enzymatic activities and second regions which encode scaffolding amino acid sequences, which method comprises modifying at least one said first region.  
     
     
         6 . The method of  claim 5  wherein said modifying comprises mutating at least one first region.  
     
     
         7 . The method of  claim 5  wherein said modifying comprises replacing at least one said first region with a region encoding the corresponding enzymatic activity from a different naturally occurring PKS gene or from a different region of the same naturally occurring PKS gene.  
     
     
         8 . The method of  claim 5  wherein said nucleotide sequence encodes at least three PKS modules.  
     
     
         9 . The method of  claim 5  wherein said modifying results in utilization of a different extender unit.  
     
     
         10 . The method of  claim 5  wherein said modifying results in utilization of a different starter unit.  
     
     
         11 . The method of  claim 5  wherein said modification results in a polyketide of a different chain length.  
     
     
         12 . The method of  claim 9  wherein said first region is an acyltransferase region.