Process and materials for production of glucosamine

Abstract

The present invention relates to a method and materials for producing glucosamine by fermentation of a genetically modified microorganism. Included in the present invention are genetically modified microorganisms useful in the present method for producing glucsamine, as well as recombinant nucleic acid molecules and the proteins produces by such recombinant nucleic acid molecules.

1 . A method to produce glucosamine by fermentation, comprising: 
 a) culturing in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate, a microorganism having a genetic modification in an amino sugar metabolic pathway, said amino sugar metabolic pathway selected from the group consisting of a pathway for converting glucosamine-6-phosphate into another compound, a pathway for synthesizing glucosamine-6-phosphate, a pathway for transport of glucosamine or glucosamine-6-phosphate out of said microorganism, a pathway for transport of glucosamine into said microorganism, and a pathway which competes for substrates involved in the production of glucosamine-6-phosphate;    wherein said step of culturing produces a product selected from the group consisting of glucosamine-6-phosphate and glucosamine from said microorganism; and    b) recovering said product.    
     
     
         2 . The method of  claim 1 , wherein said glucosamine-6-phosphate is intracellular and said glucosamine is extracellular, wherein said step of recovering comprises a recovering step selected from the group consisting of recovering said glucosamine-6-phosphate from said microorganism, recovering said glucosamine from said fermentation medium, and a combination thereof.  
     
     
         3 . The method of  claim 1 , wherein said product is glucosamine which is secreted into said fermentation medium by said microorganism and wherein said step of recovering comprises purification of said glucosamine from said fermentation medium.  
     
     
         4 . The method of  claim 1 , wherein said product is intracellular glucosamine-6-phosphate and said step of recovering comprises isolating said glucosamine-6-phosphate from said microorganism.  
     
     
         5 . (canceled)  
     
     
         6 . The method of  claim 1 , wherein said step of culturing comprises maintaining said source of carbon at a concentration of from about 0.5% to about 5% in said fermentation medium.  
     
     
         7 . The method of  claim 1 , wherein said step of culturing is performed at a temperature from about 28° C. to about 37° C.  
     
     
         8 . (canceled)  
     
     
         9 . The method of  claim 1 , wherein at least about 1 g/L of said product is recovered.  
     
     
         10 . The method of  claim 1 , wherein said genetic modification is a modification in a nucleic acid molecule encoding a protein selected from the group consisting of N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme II Nag , glucosamine-6-phosphate synthase, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme II Glc  of the PEP:glucose PTS, EIIM,P/III Man  of the PEP:mannose PTS, and a phosphatase.  
     
     
         11 . The method of  claim 1 , wherein said microorganism has a genetic modification that increases glucosamine-6-phosphate synthase action.  
     
     
         12 . The method of  claim 11 , wherein said genetic modification results in overexpression of glucosamine-6-phosphate synthase by said microorganism.  
     
     
         13 . The method of  claim 11 , wherein said genetic modification comprises transformation of said microorganism with a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase which has glucosamine-6-phosphate synthase enzymatic activity, wherein said recombinant nucleic acid molecule is operatively linked to a transcription control sequence.  
     
     
         14 - 18 . (canceled)  
     
     
         19 . The method of  claim 13 , wherein said recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase comprises a genetic modification which increases the action of said glucosamine-6-phosphate synthase.  
     
     
         20 . The method of  claim 19 , wherein said recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase comprises a genetic modification which reduces glucosamine-6-phosphate product inhibition of said glucosamine-6-phosphate synthase.  
     
     
         21 - 31 . (canceled)  
     
     
         32 . The method of  claim 11 , wherein said microorganism has at least one additional genetic modification in a gene encoding a protein selected from the group consisting of N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme II Nag , phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme II Glc  of the PEP:glucose PTS, and EIIM,P/III Man  of the PEP:mannose PTS, wherein said genetic modification decreases the action of said protein.  
     
     
         33 . The method of  claim 11 , wherein said microorganism has at least one additional genetic modification in a gene encoding a phosphatase, wherein said genetic modification increases the action of said phosphatase.  
     
     
         34 . The method of  claim 11 , wherein said microorganism has additional modifications in genes encoding the following proteins: N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific enzyme II Nag ; 
 wherein said genetic modification decreases the action of said proteins.    
     
     
         35 . (canceled)  
     
     
         36 . The method of  claim 1 , wherein said microorganism is selected from the group consisting of bacteria and yeast.  
     
     
         37 - 49 . (canceled)  
     
     
         50 . A microorganism for producing glucosamine by a biosynthetic process, said microorganism comprising: 
 a) a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase, said recombinant nucleic acid molecule being operatively linked to a transcription control sequence, wherein expression of said recombinant nucleic acid molecule increases the action of glucosamine-6-phosphate synthase by said microorganism; and,    b) at least one genetic modification in a gene encoding a protein selected from the group consisting of N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme II Nag , phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme II Glc  of the PEP:glucose PTS, and EIIM,P/III Man  of the PEP:mannose PTS, wherein said genetic modification decreases the action of said protein.    
     
     
         51 . The microorganism of  claim 50 , wherein said microorganism has at least one additional genetic modification in a gene encoding a phosphatase, wherein said genetic modification increases the action of said phosphatase.  
     
     
         52 - 59 . (canceled)  
     
     
         60 . A method to produce glucosamine by fermentation, comprising: 
 a) culturing in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate, a genetically modified microorganism having increased glucosamine-6-phosphate synthase action, wherein said genetically modified microorganism is produced by a process comprising the steps of: 
 (1) generating modifications in an isolated nucleic acid molecule comprising a nucleic acid sequence encoding glucosamine-6-phosphate synthase to create a plurality of modified nucleic acid sequences;  
 (2) transforming microorganisms with said modified nucleic acid sequences to produce genetically modified microorganisms;  
 (3) screening said genetically modified microorganisms for glucosamine-6-phosphate synthase action; and,  
 (4) selecting said genetically modified microorganisms which have increased glucosamine-6-phosphate synthase action;  
   wherein said step of culturing produces a product selected from the group consisting of glucosamine-6-phosphate and glucosamine from said microorganism; and,    b) recovering said product.