Fermentation process for producing monosaccharides in free form from nucleotide-activated sugars

Abstract

The present invention relates to a process for producing a monosaccharide, e.g. L-fucose, in free form using a microbial fermentation process. The used microorganism exhibits hydrolase activity on nucleotide-activated sugars and releases the monosaccharide in an unmodified free form. The free monosaccharide is retrieved from the supernatant of the cultivated microorganism.

1 . Process for producing a monosaccharide of interest in free form using a microorganism, the process comprising
 a.) providing a microorganism for the synthesis of the monosaccharide comprising an enzyme capable of catalyzing the hydrolysis of a nucleotide-activated monosaccharide to release the monosaccharide of interest from the nucleotide-activated monosaccharide, and   b.) cultivating the microorganism in a medium suitable for growing the microorganism, wherein the microorganism is unable to metabolize the monosaccharide to a significant extent, so that the monosaccharide of interest accumulates during cultivation.   
     
     
         2 . The process of  claim 1 , wherein a recombinant microorganism is used, wherein the recombinant microorganism comprises a heterologous nucleic acid encoding an enzyme capable of catalyzing the hydrolysis of a nucleotide-activated monosaccharide. 
     
     
         3 . The process of  claim 1 , wherein the enzyme is a glycosyltransferase, optionally a fucosyltransferase, the enzyme being able to catalyze the hydrolysis of the nucleotide-activated monosaccharide GDP-fucose in the absence of an acceptor molecule. 
     
     
         4 . The process of  claim 1 , wherein the enzyme is a variant of the 2-fucosyltransferase encoded by the wbgL gene from  Escherichia coli , or a variant of the 1,2-fucosyltransferase encoded by the futC gene from  Helicobacter pylori , the variant carrying at least one, optionally at least two, and optionally more than two modifications as compared to the wild type 2-fucosyltransferase encoded by the wbgL gene or to the wild type 1,2-fucosyltransferase encoded by the futC gene, respectively, the modification leading to an increased hydrolizing activity of the enzyme. 
     
     
         5 . The process of  claim 4 , wherein at least one modification is an amino acid substitution. 
     
     
         6 . The process of  claim 1 , wherein the microorganism is further modified to have inactivated or severely reduced or to lack catabolic pathways leading to the degradation of the produced monosaccharide. 
     
     
         7 . The process of  claim 1 , wherein the microorganism is further modified to have inactivated or severely reduced or to lack genes involved in the catabolism of L-fucose. 
     
     
         8 . The process of  claim 1 , wherein the microorganism is further modified to overexpress at least one gene involved in the biosynthesis of the nucleotide-activated monosaccharide to improve supply of the nucleotide-activated monosaccharide of the monosaccharide. 
     
     
         9 . The process of  claim 1 , wherein at least one gene involved in the biosynthesis of GDP-fucose, GDP-mannose or GDP-rhamnose is overexpressed to improve supply of GDP-fucose, GDP-mannose or GDP-rhamnose, respectively. 
     
     
         10 . The process of  claim 8 , wherein the at least one gene is heterologous or homologous. 
     
     
         11 . The process of  claim 1 , wherein the microorganism is further modified to have inactivated or reduced competing pathways for the nucleotide-activated monosaccharide. 
     
     
         12 . The process of  claim 1 , wherein the monosaccharide produced is selected from L-fucose, L-rhamnose, or L-mannose. 
     
     
         13 . The process of  claim 1 , wherein the microorganism is cultivated in a medium containing an inexpensive carbon source that is selected but not limited to glycerol, sucrose, glucose, fructose, molasse, xylose, cellulose, syngas, corn-syrup or lactose. 
     
     
         14 . The process of  claim 1 , wherein the microorganism is further modified to express a phosphatase, in case where the monosaccharide is released in a phosphorylated form by the enzyme. 
     
     
         15 . Recombinant microorganism comprising a heterologous enzyme capable of catalyzing the hydrolysis of a nucleotide-activated monosaccharide and releasing the monosaccharide from the nucleotide-activated monosaccharide in the absence of an acceptor molecule, wherein the enzyme is a glycosyltransferase, optionally a fucosyltransferase. 
     
     
         16 . The recombinant microorganism of  claim 15 , wherein the enzyme is a variant of the 2-fucosyltransferase encoded by the wbgL gene, or a variant of the 1,2-fucosyltransferase encoded by the futC gene from  Helicobacter pylori , the variant carrying at least one, optionally at least two, and optionally more than two modifications as compared to the wild type 2-fucosyltransferase encoded by the wbgL gene or to the wild type 1,2-fucosyltransferase encoded by the futC gene, respectively, the modification leading to an increased hydrolizing activity of the enzyme. 
     
     
         17 . The recombinant microorganism of  claim 15 , that is further modified to comprise heterologous genes encoding phosphomannomutase, mannose-1-phosphate guanosyltransferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase. 
     
     
         18 . The process of  claim 1 , wherein or recombinant host microorganism produced therefrom wherein the host microorganism is an  Escherichia coli  strain,  Corynebacterium  spp.,  Clostridium  spp.,  Bacillus  spp.  Pseudomonas  spp.  Lactobacillus  spp. or a  Saccharomyces  sp. strain.