The present invention relates to process for producing monosaccharides, in particular for producing L-fucose or other scarce monosaccharides e.g. N-acetyl neuraminic acid, whereby a microorganism is used in the production process.
Carbohydrates are necessary in all forms of life by taking on vital functions in energy storage, structural function, signalling, information storage etc. For this task nature synthesizes several major monosaccharides like glucose, mannose, fructose, fucose, ribose, sialic acid, etc. and several minor ones for more specialized applications, like for example D-allose.
Whereas some monosaccharides can be obtained from nature in large amounts and at reasonable cost (e.g. glucose and fructose), most monosacccharides are rather scarce and can be found in nature only in small amounts, like for example L-fucose (6-deoxy-L-galactose).
For commercial production of monosaccharides, almost exclusively oligosaccharides obtained from nature are used as sources. These oligosaccharides are acid hydrolyzed and from the released monosaccharides the individual sugars are purified. Due to the high chemical similarity of the monosaccharides (mostly differing from each other only by the orientation of individual hydroxyl-groups) the separation of individual monosaccharides in pure form is rather laborious and costly.
L-fucose represents such a rare sugar, which is currently obtained via the hydrolysis of complex oligosaccharides, either from algae or bacterial origin. For the purification of individual monosaccharides from complex hydrolysates often noxious chemicals have to be employed, like for example lead acetate and excessive amounts of organic solvents. Therefore, the isolation of individual monosaccharides from a complex hydrolysate of oligosaccharides is challenging (due to the high chemical similarity of the individual monosaccharides released) and environmentally harmful (due to the excessive use of toxic chemicals, such a lead carbonate). Also the availability of oligosaccharides rich in a certain sugar can be rather restricted in nature and also highly variable due to seasonal changes. L-Fucose represents such a scare monosaccharide which is traditionally obtained by the acid hydrolysis of fucose-containing polysaccharides. Fucose is mainly derived from the polysaccharide fucoidan, a fucan monosulfate present in all common brown seaweeds comprising the families Fucaceae and Laminariaceae. Today, L-fucose is obtained in large quantities manly by the collection of brown seaweed belonging to the family Fucaceae, which can be found world-wide but in high amounts at the European shores of the Atlantic Ocean. The large-scale harvest of brown seaweed from sea shores causes environmental concerns and is limited by certain environmental protection laws.
For example, JP 2000351790 discloses a method for extracting fucoidan and for obtaining and separating a fucose-containing oligosaccharide from the extracted fucoidan.
Besides the hydrolysis of fucoidan from brown-seaweed recently a patent publication showed that L-fucose can also be obtained via the hydrolysis of natural occurring L-fucose containing bacterial polysaccharides: WO 2012/034996 A1 discloses a strain belonging to the Enterobacteriaceae family, which strain is able to produce extracellular polysaccharides which contain L-fucose. For the production of L-fucose, the polysaccharides produced by the strain are recovered and subjected to hydrolysis, e.g. by treatment with sulphuric acid or hydrochloric acid.
Besides the extraction of L-fucose from poly- or oligosaccharide hydrolysates, several synthetic routes for L-fucose have been developed starting from other monosaccharides, like L-arabinose, D-galactose, L-rhamnose, D-mannose and D-glucose. Generally the yields of these chemical syntheses are often rather poor and involve several steps. Besides involving several synthetic steps, extensive protection group chemistry has to be used for the chemical synthesis of L-fucose. In general, the large-scale chemical synthesis of monosaccharides have not proved economical viable in comparison to extraction from nature.
Thus, currently, the preparation of any monosaccharide in pure form requires a significant effort in the purification of other monosaccharides away from the target monosaccharide, often involving large volumes of organic solvents and other noxious chemicals. As a consequence, the exclusive accumulation of a single desired monosaccharide like for example L-fucose would be of immense help. Most microorganisms are restricted in the kinds of monosaccharides they are able to utilize. In addition, they often exert strong preferences towards certain monosaccharides in case that several monosaccharides are available at the same time as carbon source.
USPTO Backgrounds
USPTO Backgrounds