Mutants of lactobacillus casei defective in carbon catabolism regulation

Abstract

The invention relates to the use of mutants of L. casei having at least a mutation impairing the regulation of a carbon catabolite repression (CCR) mechanism involving the PTS protein HPRr, for the preparation of a food product. The use of said mutants allows for instance to impart to said food products an improved texture and flavor, and/or a higher content in aroma compounds.

1 ) Use of a mutant of  L. casei  having at least a mutation impairing the regulation of a carbon catabolite repression (CCR) mechanism involving the PTS protein HPr, for the preparation of a food product.  
     
     
         2 ) The use of  claim 1 , wherein said mutant is selected from the group consisting of: 
 a) mutants having at least a mutation impairing the regulation of CCR through P-His-HPr;    b) mutants having at least a mutation impairing the regulation of CCR through P-Ser-HPr.    
     
     
         3 ) The use of  claim 2 , wherein said mutant has at least a mutation in a gene encoding a component of the PTS system.  
     
     
         4 ) The use of  claim 3 , wherein said mutant has at least a mutation selected in the group consisting of: 
 a mutation in the ptsH gene impairing the ability of HPr to be phosphorylated at His-15, or to phosphorylate EIIA;    a mutation in the ptsI gene impairing the ability of EI to phosphorylate HPr;    a mutation in a gene encoding an enzyme EIIA, EIIB, or EIIC impairing the transfer of a phosphory group to a carbohydrate.    
     
     
         5 ) The use of  claim 2 , wherein said mutant has at least a mutation in a gene encoding an antiterminator or a transcriptional activator having the PTS regulation domain.  
     
     
         6 ) The use of  claim 2 , wherein said mutant has at least a mutation selected in the group consisting of: 
 a mutation in the ptsH gene, impairing the ability of HprK to be phosphorylated at Ser-46;    a mutation in the hprK gene, impairing the ability of HprK to phosphorylate HPr at Ser-46;    a mutation in the ptsH gene, or in the ccpA gene, impairing the formation of a complex between CcpA and P-Ser-HPr or the binding of said complex to cre elements.    
     
     
         7 ) The use of any of  claims 1  to  6 , wherein said mutant is elected in the group consisting of: 
 mutants having at least a mutation in the ptsI gene resulting in the expression of an EI protein devoid of at least aminoacids 110 to 574 of wild-type EI.  
 mutants having at least a mutation in the hprK gene resulting in the expression of a HprK devoid of at least aminoacids 208 to 319 of wild-type HprK.  
 mutants having at least a mutation in the ccpA gene resulting in the expression of a CcpA deleted of at least aminoacids 134 to 333 of wild-type CcpA.  
 mutants in the ptsH gene having at least a mutation resulting in the expression of a HPr having at least one amino-acid substitution at position 15 and/or at position 46 and/or at position 47 of wild-type HPr, and/or at least a mutation resulting in the expression of a HPr deleted of at least one of amino-acids 15, 46, and/or 47 of wild-type HPr.  
 
     
     
         8 ) A mutant of  L. casei  having at least one mutant in at least one of ptsI, ptsH or hprK genes, wherein said mutation impairs at least one of the functions of the product of said genes.  
     
     
         9 ) A food-grade mutant of  L. casei , having at least one mutation in any of ptsI, ptsH, hprK, or ccpA genes.  
     
     
         10 ) A method for obtaining a food-grade mutant of  claim 9 , wherein said method comprises: 
 transforming  L. casei  with an integrative vector comprising a mutated gene selected among ptsI, ptsH, hprK, or ccpA, and further comprising a selective marker gene;    culturing the bacteria under selective conditions for the marker gene in order to obtain the bacteria having integrated the plasmid into their chromosome by a single recombination event;    culturing said bacteria under non-selective conditions for the marker gene in order to obtain bacteria having undergone a double recombination event leading to the excision of the vector sequences.    
     
     
         11 ) A method for obtaining a food-grade mutant of  L. casei  wherein the catalytic function of HPr is only slightly impaired wherein said method comprises: 
 providing a mutant strain of  L. casei  wherein the ptsI gene is inactivated in such a way that the function of EI is totally impaired;    transforming said mutant strain with an integrative vector comprising a ptsHI operon consisting of a wild type ptsI gene and the mutant ptsH gene, and further comprising a selective marker gene;    culturing the bacteria on lactose under selective conditions for the marker gene, and recovering the bacteria having integrated the vector into their chromosome by a single recombination event;    culturing the selected bacteria on lactose and under non-selective conditions for the marker gene in order to obtain bacteria having undergone a double recombination event leading to the excision of the vector sequences.    
     
     
         12 ) A process for preparing a food product or food additive wherein said process comprises fermenting a food substrate with a mutant of  L. casei , as defined in any of  claims 1  to  9 .  
     
     
         13 ) A process of  claim 12  wherein said food product is a dairy product.  
     
     
         14 ) A process of any of claims  12  or  13 , for preparing a food product enriched with aroma compounds, comprising by fermenting a food substrate with a strain of L. casei  having a mutation impairing the function of CcpA.  
     
     
         15 ) A process of any of claims  12  or  13 , for preparing a food product having an improved texture and flavor, comprising fermenting a food substrate with a strain of  L. casei  having a mutation impairing the function of EI.  
     
     
         16 ) A fermented food product obtainable by a process according to any of claims  12  to 15.  
     
     
         17 ) A fermented food product comprising at least a mutant of  L. casei , as defined in any of  claims 1  to  9 .