Methods and compositions for genetically manipulating clostridia and related bacteria with homologous recombination associated proteins

Abstract

Methods for effecting homologous recombination in a bacterium of the Clostridia family are described. These methods provide enhanced capability to genetically modify clostridia.

1 . A method for effecting an efficient single-crossover or double-crossover homologous recombination in a population of bacteria comprising singly or combinatorially expressing a recombination protein selected from the group consisting of RecG, RecO and RecA, wherein recombinant bacteria are produced. 
     
     
         2 . The method of  claim 1 , wherein the homologous recombination proteins are derived from any RecG, RecO, or RecA gene or variant capable of functioning in the recombinant bacteria. 
     
     
         3 . The method of  claim 1 , wherein functional RecG, RecO, RecA and combinations thereof are expressed by the recombinant bacteria. 
     
     
         4 . A recombinant bacterium produced by the method of  claim 1 . 
     
     
         5 . The method of  claim 1 , wherein the genetic heterogeneity of the population of recombinant bacteria is increased. 
     
     
         6 . The method of  claim 1  further comprising screening the recombinant bacteria for desired phenotypic traits. 
     
     
         7 . A method for integrating a recombinant gene into a host chromosome for recombinant protein expression comprising the method of  claim 1 . 
     
     
         8 . The method of  claim 7 , wherein the integrated recombinant gene is a synthetic operon. 
     
     
         9 . The method of  claim 7 , wherein expression of a gene is suppressed. 
     
     
         10 . The method of  claim 7 , wherein expression of a gene is stimulated. 
     
     
         11 . The method of  claim 7 , wherein the timing of expression of a gene is altered. 
     
     
         12 . The method of  claim 7 , wherein expression of a first gene is repressed by the expression of a second gene. 
     
     
         13 . The method of  claim 7 , wherein expression of a first gene is induced by the expression of a second gene. 
     
     
         14 . The bacterium of  claim 4 , wherein the bacterium is a Clostridial bacterium.