Process for producing monosaccharides
The present invention relates to a process for producing a monosaccharide using a microorganism. The microorganism possesses a glycosyltransferase and a glycosidase which work together to synthesize a desired monosaccharide in free form by using an endogenous provided nucleotide activated monosaccharide, glycosylate a suitable acceptor substrate and release the desired monosaccharide by a hydrolysis reaction. The required acceptor substrate for the reaction is recycled and only needed in catalytic amounts. The monosaccharide is produced in free from and is retrieved from the supernatant of the cultivated microorganism.
1. A process for producing L-fucose in free form using a bacterial microorganism, the process comprising the steps of:
a) providing a recombinant bacterial microorganism that has been transformed to contain a nucleic acid sequence encoding a bacterial alpha-1,2-fucosyltransferase specifically able to transfer fucose from an activated nucleotide fucose to lactose to form a lactose-fucose substrate, or to lactulose to form a lactulose-fuscose substrate, and a nucleic acid sequence encoding a bacterial 1,2-alpha-fucosidase not naturally occurring in the recombinant bacterial microorganism, the bacterial 1,2-alpha-fucosidase able to release the L-fucose from the lactose-fucose substrate or from the lactulose-fucose substrate, wherein the recombinant bacterial microorganism lacks a beta-galactosidase gene or comprises a deregulated beta-galactosidase-encoding gene, and wherein the recombinant bacterial microorganism is unable to metabolize L-fucose;
b) cultivating the recombinant bacterial microorganism in a medium suitable for growing the recombinant bacterial microorganism, whereby L-fucose is produced in a free form; and
c) recovering the L-fucose from the medium.
2. The process of claim 1 , wherein the medium comprises a carbon source, and wherein the carbon source is glycerol, sucrose, molasses, xylose, cellulose, or syngas.
3. The process of claim 1 , wherein cultivating the recombinant bacterial microorganism is a batch or a continuous process.
4. The process of claim 1 , wherein recovering free L-fucose from the medium comprises centrifuging the cultivated recombinant bacterial microorganism to obtain a supernatant and a recombinant bacterial microorganism pellet, and recovering the free L-fucose from the supernatant.
5. A process for producing L-fucose in free form using a bacterial microorganism, the process comprising the steps of:
a) providing a recombinant E. coli host cell that has been transformed to contain i) a wbgL gene encoding a bacterial alpha 1,2-fucosyltransferase, wherein the alpha 1,2-fucosyltransferase can transfer the fucose from an activated nucleotide fucose to lactose or lactulose to form a lactose-fucose-substrate or a lactulose-fucose-substrate, and ii) an afcA gene encoding a 1,2-alpha-fucosidase not naturally occurring in the recombinant E. coli host cell, wherein the 1,2-alpha-fucosidase can release L-fucose from the lactose-fucose-substrate or the lactulose-fucose-substrate; wherein the recombinant E. coli host cell is unable to metabolize the L-fucose;
b) cultivating the recombinant E. coli host cell in a medium suitable for growing the recombinant E. coli host cell, whereby L-fucose is produced in a free form; and
c) recovering the L-fucose from the medium.
6. The process of claim 5 , wherein the recombinant E. coli host cell is further transformed to contain the gal-operon (galETKM) genes and the lactose transporter gene lacY.
7. The process of claim 6 , wherein the medium comprises a carbon source, and wherein the carbon source is glycerol, sucrose, molasses, xylose, cellulose, or syngas.
8. The process of claim 6 , wherein cultivating the recombinant E. coli is a batch or a continuous process.
9. The process of claim 6 , wherein recovering free L-fucose from the medium comprises centrifuging the cultivated E. coli host cell to obtain a supernatant and a E. coli host cell pellet, and recovering the free L-fucose from the supernatant.
10. The process of claim 1 , wherein the recombinant bacterial microorganism is a recombinant Escherichia coli.
11. The process of claim 1 , wherein the alpha-1,2-fucosyltransferase is encoded by a wbgL gene of E. coli.
12. The process of claim 1 , wherein the 1,2-alpha-glycosidase is encoded by an afcA gene of Bifidobacterium bifidum.