Directed evolution of microorganisms

Abstract

The present invention provides methods for directing the evolution of microorganisms comprising the use of mutator genes and growth under conditions of selective pressure. The method discloses mutator genes which can be used in the methods of the present invention and provides ATCC deposits which exemplify the evolved microorganisms produced by the methods.

1 . A method for preparing an evolved microorganism comprising the steps of:
 a. culturing a microorganism comprising at least one heterologous mutator gene for at least 20 doublings under conditions suitable for selection of an evolved microorganism, wherein said heterologous mutator gene generates a mutation rate of at least 5-100,000 fold relative to wild type, and   b. restoring said evolved microorganism to a wild type mutation rate.   
     
     
         2 . The method of  claim 1  wherein said microorganism further comprises at least one introduced nucleic acid encoding a heterologous protein. 
     
     
         3 . The method of  claim 2  wherein said heterologous protein(s) includes hormones, enzymes and growth factors. 
     
     
         4 . The method of  claim 3  wherein said heterologous protein is an enzyme. 
     
     
         5 . The method of  claim 4  wherein said enzyme includes hydrolases, such as protease, esterase, lipase, phenol oxidase, permease, amylase, pullulanase, cellulase, glucose isomerase, laccase and protein disulfide isomerase. 
     
     
         6 . The method of  claim 1  wherein said microorganism further comprises introduced nucleic acid encoding at least one enzyme necessary for an enzymatic pathway. 
     
     
         7 . The method of  claim 6  wherein said enzyme is a reductase or a dehydrogenase and said enzymatic pathway is for the production of ascorbic acid or ascorbic acid intermediates. 
     
     
         8 . The method of  claim 6  wherein said enzyme is glycerol dehydratase or 1,3-propanediol dehydrogenase and said enzymatic pathway is for the production of 1,3 propanediol, 1,3 propanediol precursors or 1,3 propanediol derivatives. 
     
     
         9 . The method of  claim 6  wherein said enzyme is glycerol-3-phosphate dehydrogenase or glycerol-3-phosphate phosphatase and said pathway is for the production of glycerol and glycerol derivatives. 
     
     
         10 . The method of  claim 6  wherein said enzymatic pathway is for the production of amino acids or dyes. 
     
     
         11 . The method of  claim 1  wherein said microorganism is cultured for between about 20 to about 100 doublings. 
     
     
         12 . The method of  claim 1  wherein said microorganism is cultured for between about 100 to about 500 doublings. 
     
     
         13 . The method of  claim 1  wherein said microorganism is cultured for between about 500 to about 2000 doublings. 
     
     
         14 . The method of  claim 1  wherein said microorganism is cultured for greater than 2000 doublings. 
     
     
         15 . The method of  claim 1  wherein said evolved microorganism comprises from about 3 to about 1000 selected mutations. 
     
     
         16 . The method of  claim 1  wherein said evolved microorganism further comprises from about 20 to about 100,000 neutral mutations 
     
     
         17 . The method of  claim 1  wherein said evolved microorganism comprises about 3 to about 1000 selected mutations in about 3 to about 500 genes. 
     
     
         18 . The method of  claim 17  wherein said mutations are non-specific. 
     
     
         19 . The method of  claim 17  wherein said mutations are specific. 
     
     
         20 . The method of  claim 1  wherein said mutator gene generates a mutation rate of at least about 5 fold to about 10,000 fold relative to wild type. 
     
     
         21 . The method of  claim 1  wherein said mutator gene generates a mutation rate of at least about 5 fold to about 1000 fold. 
     
     
         22 . The method of  claim 1  wherein said mutator gene generates a mutation rate of about 5 fold to about 1000 fold over wild type. 
     
     
         23 . The method of  claim 1  wherein said microorganism comprises a plasmid comprising the heterologous mutator gene and said step of restoring said evolved microorganism to a wild type mutation rate comprises curing the evolved microorganism of said plasmid. 
     
     
         24 . The method of  claim 23  wherein said plasmid comprises a temperature sensitive origin of replication. 
     
     
         25 . The method of  claim 1  wherein said microorganism comprises at least one copy of the mutator gene in the chromosome and said step of restoring said evolved microorganism to wild type mutation rate comprise excision of said mutator gene. 
     
     
         26 . The method of  claim 1  wherein said mutator gene comprises mutD, mutT, mutY, mutM, mutH, mutL, mutS or mutU mutations or homologues thereof. 
     
     
         27 . The method of  claim 26  wherein said mutator gene comprises mutD having mutations shown in Table I. 
     
     
         28 . The method of  claim 1  wherein said conditions suitable for selection comprise culturing said microorganism in the presence of at least one organic solvent. 
     
     
         29 . The method of  claim 28  wherein said organic solvent includes alcohols, diols, hydrocarbon, mineral oil, mineral oil derived products, halogenated compounds and aromatic compounds. 
     
     
         30 . The method of  claim 1  wherein said conditions suitable for selection comprise culturing said microorganism in the presence of elevated temperature. 
     
     
         31 . The method of  claim 30  wherein said elevated temperature is about 42° C. to about 48° C. 
     
     
         32 . The method of  claim 1  wherein said conditions suitable for selection comprise culturing said microorganism in the presence of high salt. 
     
     
         33 . The method of  claim 1  wherein said microorganism includes Gram-positive or a Gram-negative microorganism, fungus, yeast or eucaryotic. 
     
     
         34 . The method of  claim 33  wherein said microorganism is an Enterobacteriaceae. 
     
     
         35 . The method of  claim 34  wherein said microorganism is an  Eschericia.    
     
     
         36 . The method of  claim 35  wherein said microorganism is  E. coli.    
     
     
         37 . The method of  claim 35  wherein said microorganism is  E. blatte.    
     
     
         38 . The method of  claim 1  wherein said evolved microorganism is  E. coli  having ATCC accession number ______. 
     
     
         39 . The method of  claim 1  wherein said evolved microorganism is  E. blattae  having ATCC accession number. 
     
     
         40 . An expression vector comprising a mutator gene. 
     
     
         41 . The expression vector of  claim 40  wherein said mutator gene is a mutated MutD. 
     
     
         42 . The expression vector of  claim 40  wherein said mutated MutD has the mutations as shown in Table I. 
     
     
         43 . A host cell comprising the expression vector of  claim 40 . 
     
     
         44 . The host cell of  claim 43  that is a Gram-positive or Gram-negative microorganism. 
     
     
         45 . The host cell of  claim 44  that is an Enterobacteriaceae. 
     
     
         46 . The isolated  E. blattae  microorganism deposited with the ATCC and having accession number. 
     
     
         47 . The isolated  E. coli  microorganism deposited with the ATCC and having accession number. 
     
     
         48 . A method for preparing an evolved microorganism comprising the steps of:
 a. mutating a DNA repair gene in a microorganism to obtain a mutated strain,   b. culturing the mutated strain for at least 20 doublings under conditions suitable for selection of an evolved strain, wherein said mutated strain generates a mutation rate of at least 5-100,000 fold relative to the wild-type microorganism, and   c. restoring the naturally occurring DNA repair gene in said evolved microorganism.