Production of oligosaccharides
The present invention relates to the use of one or more glycosidases in the process for the production and/or purification of a produced desired oligosaccharide. The process is preferably a microbial fermentation process using a host microorganism, which may also comprise nucleic acids expressing sugar catabolic pathway proteins suitable for the degradation of saccharides otherwise hindering the purification of the desired oligosaccharide.
1. A process for producing a desired oligosaccharide in a host microorganism, wherein the desired oligosaccharide is lacto-N-tetraose and is not naturally occurring in said host microorganism, the process comprising:
a) cultivating a host microorganism suitable for the production of the desired oligosaccharide under conditions and in a medium permissive for the production of the desired oligosaccharide, thereby producing the desired oligosaccharide and undesired biosynthetic saccharide intermediates and/or unused saccharide side products;
b) adding a glycosidase to the medium at the end of the cultivation of a) to degrade undesired biosynthetic saccharide intermediates and/or unused saccharide side products and/or unused saccharide substrates, wherein the glycosidase is added by adding a second microorganism to the medium which expresses the glycosidase, wherein said glycosidase is a beta-galactosidase and a beta-N-acetylhexosaminidase, or a beta-galactosidase and a beta-N-acetylglucosaminidase; and
c) recovering the desired oligosaccharide.
2. The process of claim 1 , wherein the process is a batch or a continuous process.
3. The process of claim 1 , wherein the desired oligosaccharide is recovered from supernatant of the cultivated host microorganism, which supernatant is obtained by centrifuging the cultivated host microorganism to obtain a supernatant and a host microorganism pellet.
4. The process of claim 1 , wherein the host microorganism is a bacterium or a yeast.
5. The process of claim 1 , wherein the host microorganism is an Escherichia coli strain, Lactobacillus species, a Corynebacterium glutamicum strain, or a Saccharomyces sp. strain.
6. The process of claim 1 , wherein the second microorganism employed expresses proteins for sugar catabolic pathways otherwise not present in the microorganism, wherein the proteins comprise at least one of:
a) galactose catabolic pathway proteins when the glycosidase is a galactosidase;
b) and
N-acetylglucosamine catabolic pathway proteins.
7. The process of claim 1 , wherein the host microorganism overexpresses a salvage pathway for a monosaccharide generated during production of the desired oligosaccharide, to convert the monosaccharide into its nucleotide-activated form.
8. The process of claim 1 , wherein the host microorganism employed is a wild-type microorganism modified to express proteins for sugar catabolic pathways under the control of an inducible promoter, wherein the sugar catabolic proteins are: galactose catabolic pathway proteins, and/or N-acetylglucosamine catabolic pathway proteins.
9. The process of claim 1 , wherein the N-acetylhexosaminidase is encoded by the Bifidobacterium bifidum JCM1254 bbhI gene.
10. The process of claim 1 , wherein the glycosidase is encoded by a nucleic acid sequence that has been stably integrated into the genome of the second microorganism.
11. The process of claim 1 , wherein the undesired biosynthetic saccharide intermediates comprises lacto-N-triose II.
12. The process of claim 1 , wherein the unused saccharide side products comprises lactose.
13. The process of claim 2 , wherein the process is a continuous process which employs two separate fermentation vessels, wherein one fermentation vessel is used for the oligosaccharide synthesis, and the second fermentation vessel is employed for the degradation of the undesired biosynthetic saccharide intermediates and/or unused saccharide side products.