FERMENTATION PROCESS FOR PRODUCING MONOSACCHARIDES IN FREE FORM FROM NUCLEOTIDE-ACTIVATED SUGARS
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.