Methods for the production of products in host cells

Abstract

The invention provides methods and host cells for the production of ascorbic acid intermediates. The invention also provides host cells having a modification in a polynucleotide that uncouples the catabolic pathway from the oxidative pathway by deleting the encoding for an endogenous enzymatic activity that phosphorylates D-glucose at its 6th carbon and/or a polynucleotide that has deleted the encoding for endogenous enzymatic activity that phosphorylates D-gluconate at its 6th carbon. Such host cells are used for the production of products, such as, ascorbic acid intermediates. Nucleic acid and amino acid sequences with inactivated enzymatic activity which phosphorylates D-glucose at its 6th carbon and inactivated enzymatic activity which phosphorylates D-gluconate at its 6th carbon are provided.

1 . A process for producing an ascorbic acid intermediate in a recombinant host cell comprising, culturing a host cell capable of producing said ascorbic acid intermediate in the presence of glucose under conditions suitable for the production of said ascorbic acid intermediate wherein said host cell comprises at least one deletion selected from the group of polynucleotides encoding kinases from the group consisting of glucokinase and gluconokinase.  
     
     
         2 . The process of  claim 1  wherein said host cell further comprises both deletions selected from the group of residues encoding kinases selected from the group consisting of glucokinase and gluconokinase.  
     
     
         3 . The process of  claim 1  further comprising the step of recovering said product.  
     
     
         4 . The process of  claim 1  further comprising the step of converting ascorbic acid intermediate into a second product.  
     
     
         5 . A method of enhancing the production of an ascorbic acid intermediate from a carbon source comprising, 
 d) obtaining an altered  Pantoea  bacterial strain which comprises inactivating a chromosomal gene in a bacterial host strain,    e) culturing said altered bacterial host strain under suitable conditions, and    f) allowing the production of an ascorbic acid intermediate from the carbon source, wherein the production of said ascorbic acid intermediate is enhanced compared to the production of the ascorbic acid intermediate in the unaltered bacterial host strain.    
     
     
         6 . The method of  claim 5 , wherein the ascorbic acid intermediate is selected from the group consisting of gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, L-iodonic acid, erythorbic acid, and tartrate.  
     
     
         7 . The method of  claim 6 , wherein the ascorbic acid intermediate is 2,5-diketo-D-gluconate.  
     
     
         8 . The method of  claim 5 , wherein said altered bacterial strain is obtained by the inactivation of a glk chromosomal gene.  
     
     
         9 . The method of  claim 5 , wherein said altered bacterial strain is obtained by the inactivation of a gntk chromosomal gene.  
     
     
         10 . The method of  claim 5 , wherein said altered bacterial strain is obtained by the inactivation of a glk and a gntk chromosomal gene.  
     
     
         11 . The method of  claim 5 , wherein said bacterial strain is  Pantoea.    
     
     
         12 . The method of  claim 11 , wherein said bacterial strain is  Pantoea citrea. GC 620-3 50