Enterobacteriaceae fermentation strains

Abstract

Methods are provided for preparing improved fermentation strains of the family Enterobacteriaceae which comprise the steps of eliminating the cryptic plasmid from the progenitor strain thereby creating the improved strain. Methods for reducing the mobilization properties of resident plasmids in an Enterobacteriaceae strain containing a cryptic plasmid are also provided. The present invention provides the nucleic acid sequence of pS, a cryptic plasmid found in Pantoea which can be used to identify the cryptic plasmid in strains of Enterobacteriaceae.

1 - 19 . (canceled)  
     
     
         20 . An isolated protein having the amino acid sequence as shown in SEQ ID NO: 3.  
     
     
         21 . A method for preparing an improved Enterobacteriaceae strain from a progenitor strain comprising, 
 a) obtaining a progenitor strain selected from the group consisting of  Pantoea, Enterobacter, Erwinia  and  Gluconobacter,  wherein the progenitor strain contains a cryptic plasmid which comprises a nucleic acid sequence which encodes an amino acid having at least 95% sequence identity with SEQ ID NO: 3;    b) eliminating said cryptic plasmid from the progenitor strain and    c) obtaining an improved Enterobacteriaceae strain.    
     
     
         22 . The method according to  claim 21 , wherein the improved strain is a  Pantoea  strain.  
     
     
         23 . The method according to  claim 22 , wherein the  Pantoea  strain is a  P. citrea.    
     
     
         24 . The method according to  claim 21 , wherein the progenitor strain and the improved strain are capable of producing 2,5-diketo-D-gluconate from a carbon source.  
     
     
         25 . The method according to  claim 21 , wherein the progenitor strain is a recombinant strain that comprises a heterologous nucleic acid sequence encoding a 2,5,-diketo-D-gluconate reductase and is capable of converting 2,5-diketo-D-gluconate to 2-keto-L-gluconic acid.  
     
     
         26 . The method according to  claim 21 , wherein the cryptic plasmid has the nucleic acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2.  
     
     
         27 . The method according to  claim 21 , wherein the cryptic plasmid comprises a nucleic acid sequence which encodes an amino acid having the sequence of SEQ ID NO: 3.  
     
     
         28 . The method according to  claim 21 , wherein mobilization properties of plasmids residing within the progenitor strain are reduced in the improved strain.  
     
     
         29 . The method according to  claim 21 , wherein the improved  Pantoea  strain is able to grow at a higher temperature than the progenitor strain.  
     
     
         30 . The method according to  claim 21 , wherein the cryptic plasmid has been eliminated by chemical means.  
     
     
         31 . The method according to  claim 21 , wherein the cryptic plasmid has been eliminated by recombinant means.  
     
     
         32 . The method according to  claim 21 , wherein the progenitor strain is a  Pantoea  strain, the cryptic plasmid comprises a nucleic acid sequence which encodes the amino acid sequence of SEQ ID NO: 3 and the progenitor strain and the improved strain are capable of producing 2,5-diketo-D-gluconate from a carbon source.