Methods for altering acetic acid production and enhancing cell death in bacteria

Abstract

Methods of increasing cultured cell growth yields and/or protein production from bacterial cell cultures are provided. More particularly, mutant bacterial cells having an alteration in the expression or activity of the cidABC operon, a gene therein, or a homolog or a regulator thereof, and methods for reducing acetic acid/acetate production in cultures are provided, as are methods for increasing cultured cell growth yields and/or protein production employing such cells. Methods for enhancing bacterial cell death and methods for identifying agents that increase the susceptibility of bacteria to cell death are also provided.

1 . An isolated bacterial cell, comprising a mutation in a cidABC operon homolog, wherein the mutation measurably reduces acetate production by the cell when the cell is cultured in medium comprising a metabolizable carbon source, in comparison to an isogenic cell not comprising the mutation and cultured under identical conditions to the isolated cell. 
     
     
         2 . The isolated cell of  claim 1 , wherein the mutation is an inactivating mutation in a cidC homolog encoding a pyruvate oxidase. 
     
     
         3 . The isolated cell of  claim 1 , wherein the isolated cell is of a gram positive species. 
     
     
         4 . The isolated cell of  claim 1 , wherein the isolated cell is of a gram negative species. 
     
     
         5 . The isolated cell of  claim 4 , wherein the gram negative species is  E. coli.    
     
     
         6 . The isolated cell of  claim 2 , wherein the homolog of cidC is poxB. 
     
     
         7 . The isolated cell of  claim 1 , wherein the mutation is a knockout mutation. 
     
     
         8 . A method of increasing yield in a bacterial cell culture, comprising:
 culturing the isolated bacterial cell of  claim 1 ,   
       wherein the reduced acetate production results in increased yield, and wherein increased yield is measured as an increase in bacterial biomass, an increase in production of a product, an increase in turbidity, an increase in optical density, or a combination of two or more thereof. 
     
     
         9 . The method of  claim 8 , wherein the mutation is an inactivating mutation in a cidC homolog encoding a pyruvate oxidase. 
     
     
         10 . The method of  claim 8 , wherein the isolated cell is of a gram positive species. 
     
     
         11 . The method of  claim 8 , wherein the isolated cell is of a gram negative species. 
     
     
         12 . The method of  claim 11 , wherein the gram negative species is  E. coli.    
     
     
         13 . The method of  claim 9 , wherein the homolog of cidC is poxB. 
     
     
         14 . The method of  claim 8 , wherein the mutation is a knockout mutation. 
     
     
         15 . A method of producing a polypeptide, comprising:
 culturing the isolated cell of  claim 1  in culture medium to produce a bacterial cell culture, wherein the isolated cell comprises a nucleic acid encoding the polypeptide; and   isolating the polypeptide from the bacterial cell culture.   
     
     
         16 . The method of  claim 15 , wherein the mutation is an inactivating mutation in a cidC homolog encoding a pyruvate oxidase. 
     
     
         17 . The method of  claim 15 , wherein the isolated cell is of a gram positive species. 
     
     
         18 . The method of  claim 15 , wherein the isolated cell is of a gram negative species. 
     
     
         19 . The method of  claim 18 , wherein the gram negative species is  E. coli.    
     
     
         20 . The method of  claim 16 , wherein the cidC homolog is poxB.