Production of complex carbohydrates

Abstract

Compositions and methods for making complex carbohydrates in a bacterial production cell are disclosed. The complex carbohydrates that can be made include oligosaccharides and polysaccharides of bacterial or mammalian origin.

1 - 29 . (canceled)  
   
   
       30 . A process for the production of a  Haemophilus influenzae -specific lipooligosaccharide (LOS) which comprises the steps of: 
 (a) growing in a culture medium gram-negative bacteria comprising (i) a core lipid structure containing a terminal heptose and (ii) a DNA sequence encoding an Undecaprenyl-phosphate N-acetyl glucosaminyl phosphate transferase (rfe), and (iii) an isolated DNA sequence encoding a lipooligosaccharide-synthesis gene G polypeptide (LsgG) from  Haemophilus influenzae,  wherein the DNA sequence encoding rfe is regulated by LsgG such that a  H. influenzae -specific LOS is synthesized by the addition of an acceptor molecule to the terminal heptose molecule; and    (b) recovering the  H. influenzae -specific LOS from the culture medium.    
   
   
       31 . The process of  claim 30 , wherein the bacteria are  Escherichia coli.    
   
   
       32 . The process of  claim 31 , wherein the bacteria are  Escherichia coli  K-12 strain JM 109.  
   
   
       33 . The process of  claim 30 , wherein the bacteria are  Salmonella minnesota.    
   
   
       34 . The process of  claim 30 , wherein the acceptor molecule is N-acetylglucosanine.  
   
   
       35 . The process of  claim 30 , wherein the DNA sequence encoding rfe is from  Haemophilus influenzae.    
   
   
       36 . The process of  claim 30 , wherein the DNA sequence encoding a rfe is part of the gram-negative bacterial genome.  
   
   
       37 . The process of  claim 30 , wherein the isolated DNA sequence encoding the LsgG is comprised in a vector.  
   
   
       38 . The process of  claim 30 , wherein the bacteria further comprise a glycosyltransferase.  
   
   
       39 . A process for the production of a complex carbohydrate comprising the steps of: 
 (a) growing in a culture medium gram-negative bacteria comprising (i) a core lipid structure containing a terminal heptose and (ii) a DNA sequence encoding an Undecaprenyl phosphate N-acetyl glucosaminyl phosphate transferase (rfe), and (iii) an isolated DNA sequence encoding a liposaccharide-synthesis gene G polypeptide (LsgG) from  Haemophilus influenzae,  wherein the DNA sequence encoding rfe is regulated by LsgG such that a complex carbohydrate is synthesized by the addition of an acceptor molecule to the heptose molecule; and    (b) recovering the complex carbohydrate from the culture medium.    
   
   
       40 . The process of  claim 39 , wherein the bacteria are  Escherichia coli.    
   
   
       41 . The process of  claim 40 , wherein the bacteria are  Escherichia coli  K-12 strain JM 109.  
   
   
       42 . The process of  claim 39 , wherein the bacteria are  Salmonella minnesota.    
   
   
       43 . The process of  claim 39 , wherein the acceptor molecule is N-acetylglucosamine.  
   
   
       44 . The process of  claim 39 , wherein the DNA sequence encoding rfe is from  Haemophilus influenzae.    
   
   
       45 . The process of  claim 39 , wherein the DNA sequence encoding a rfe is part of the gram-negative bacterial genome.  
   
   
       46 . The process of  claim 39 , wherein the isolated DNA sequence encoding LsgG is contained in a vector.  
   
   
       47 . The process of  claim 39 , wherein the bacteria further comprise a glycosyltransferase.  
   
   
       48 . A method of modifying a terminal heptose of a lipopolysaccharide (LPS) or lipooligosaccharide (LOS) core structure of a gram-negative bacterial species comprising a polynucleotide encoding an Undecaprenyl phosphate N-acetyl glucosaminyl phosphate transferase (rfe), wherein the polynucleotide encoding rfe is regulated by lipooligosaccharide-synthesis gene G polypeptide (LsgG) from  Haemophilus influenzae  such that an N-acetyl glucosamine is added onto the terminal heptose.  
   
   
       49 . The method of  claim 48  wherein the bacteria are  Escherichia coli.    
   
   
       50 . The method of  claim 49 , wherein the bacteria are  Escherichia coli  K-12 strain JM 109.  
   
   
       51 . The method of  claim 48 , wherein the bacteria are  Salmonella minnesota.    
   
   
       52 . The method of  claim 48 , wherein the polynucleotide encoding rfe is from  Haemophilus influenzae.    
   
   
       53 . The method of  claim 48 , wherein the polynucleotide encoding rfe is part of the gram-negative bacterial genome.  
   
   
       54 . The method of  claim 48 , wherein a polynucleotide encoding the LsgG is comprised in a vector.  
   
   
       55 . The method of  claim 48 , wherein the bacteria further comprise a glycosyltransferase.