Fermentative Production of Oligosaccharides by Microbial Cells Utilizing Glycerol
AssigneeChr. Hansen A/S
InventorsAndré Luis RODRIGUES, Christian TROETSCHEL, Henning FRERIGMANN, Markus ENGLERT, Elisabeth STAUDT-GRUNDMANN
Life of this patent
Estimating the term…
Abstract
Disclosed are methylglyoxal resistant genetically engineered microbial cells for the fermentative production of an oligosaccharide of interest, and methods for fermentative production of an oligosaccharide of interest using the methylglyoxal resistant microbial cells.
Claims14
A genetically engineered microbial cell for intracellular biosynthesis of an oligosaccharide of interest, wherein the genetically engineered microbial cell synthesizes the oligosaccharide of interest intracellularly when cultivated in the presence of glycerol as sole carbon and energy source, wherein the genetically engineered microbial cell comprises
- a glycerol permease for internalization of exogenous glycerol;
- a glycerol kinase, wherein said glycerol kinase is a functional variant of the E. coli glycerol kinase GlpK, wherein said functional variant possesses an amino acid residue at the position which corresponds to amino acid position 55 in the amino acid sequence of E. coli GlpK as set forth in SEQ ID NO:
- 1, wherein said amino acid residue comprises a non-ionized but polar acting side chain, and/or an amino acid residue at the position which corresponds to amino acid position 231 in the amino acid sequence of E. coli GlpK as set forth in SEQ ID NO:
- 1, wherein said anionic amino acid residue comprises an anionic side chain;
- a metabolic pathway for the intracellular biosynthesis of a nucleotide-activated monosaccharide as a donor substrate of the monosaccharide moiety;
- and
- a glycosyltransferase for the transfer of the monosaccharide moiety from the nucleotide-activated monosaccharide to an acceptor substrate.
9-10. (canceled)
A method for alleviating methyl glyoxal toxicity of a microbial cell, wherein the method comprises:
- providing a genetically engineered microbial cell for the fermentative production of an oligosaccharide of interest;
- deleting or functionally inactivating the endogenous glycerol kinase gene(s) of the microbial cell;
- transforming the microbial cell to contain and express a gene which encodes a functional variant of the E. coli (K12) glycerol kinase GlpK, wherein said functional variant possesses an amino acid residue at the position which corresponds to amino acid position 55 in amino acid sequence of E. coli GlpK as set forth in SEQ ID NO:
- 1, wherein said amino acid residue comprises a non-ionized but polar acting side chain, and/or an amino acid residue at the position which corresponds to amino acid position 231 in the amino acid sequence of E. coli GlpK as set forth in SEQ ID NO:
- 1, wherein said anionic amino acid residue comprises an anionic side chain.
A method for decreasing the responsivity of a microbial cell to methylglyoxal toxicity during fermentative production of an oligosaccharide of interest, wherein the method comprises:
- providing a genetically engineered microbial cell for the fermentative production of an oligosaccharide of interest;
- deleting or functionally inactivating the endogenous glycerol kinase gene(s) of the microbial cell;
- transforming the microbial cell to contain and express a gene which encodes a functional variant of the E. coli glycerol kinase GlpK, wherein said functional variant possesses an amino acid residue at the position which corresponds to amino acid position 55 in amino acid sequence of E. coli GlpK as set forth in SEQ ID NO:
- 1, wherein said amino acid residue comprises a non-ionized but polar acting side chain, and/or an amino acid residue at the position which corresponds to amino acid position 231 in the amino acid sequence of E. coli GlpK as set forth in SEQ ID NO:
- 1, wherein said anionic amino acid residue comprises an anionic side chain.
Dependencies are inferred from the claim text.