HMO production
The present inventive concept relates to a genetically modified cell enabled for the production of an oligosaccharide, preferably, an HMO, comprising a recombinant nucleic acid encoding a protein of the MFS superfamily; and methods using said cell for the production the oligosaccharide, preferably an HMO.
1 . A genetically modified cell capable of producing one or more Human Milk Oligosaccharides (HMOs) under fermentative conditions, wherein the cell comprises a recombinant nucleic acid encoding a protein of SEQ ID NO: 1, or a functional homologue thereof which amino acid sequence is at least 90% identical to SEQ ID NO: 1,
wherein the functional homologue increases the total production of the one or more HMOs while reducing by-product formation or facilitates the export of the one or more HMOs out of the genetically modified cell,
wherein the one or more HMOs are selected from the group consisting of: 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), Lacto-N-Triose-2 (LNT-2), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), Lacto-N-fucopentaose I (LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto-N-fucopentaose III (LNFP III), Lacto-N-fucopentaose IV (LNFP IV), Lacto-N-fucopentaose V (LNFP V), para-lacto-N-neohexaose (pLNnH), and para-lacto-neohexaose II (pLNH-II).
2 . The genetically modified cell of claim 1 , wherein the one or more HMOs is a trisaccharide selected from the group consisting of LNT-2, 3′-SL and 6′-SL, or a tetrasaccharide selected from the group consisting of LNT, LNnT, and FSL.
3 . The genetically modified cell of claim 2 , wherein the one or more HMOs comprises 3′-SL or 6′-SL.
4 . The genetically modified cell of claim 2 , wherein the one or more HMOs comprises LNT, LNnT, or FSL.
5 . The genetically modified cell of claim 1 , wherein the genetically modified cell is Escherichia coli.
6 . The genetically modified cell of claim 1 , wherein the cell further comprises an expression element comprising a lac promoter or a glp promoter.
7 . The genetically modified cell of claim 6 , wherein the lac promoter, if present, is Plac and the glp promoter, if present, is PglpF.
8 . The genetically modified cell of claim 1 , wherein the recombinant nucleic acid encodes a protein of SEQ ID NO: 1.
9 . The genetically modified cell of claim 8 , wherein the one or more HMOs is a trisaccharide selected from the group consisting of LNT-2, 3′-SL and 6′-SL, or a tetrasaccharide selected from the group consisting of LNT, LNnT or FSL.
10 . The genetically modified cell of claim 9 , wherein the one or more HMOs comprises 3′-SL or 6′-SL.
11 . The genetically modified cell of claim 9 , wherein the one or more HMOs comprises LNT, LNnT, or FSL.
12 . The genetically modified cell of claim 1 , wherein the recombinant nucleic acid has at last 85% sequence identity to SEQ ID NO: 2.
13 . The genetically modified cell of claim 12 , wherein the one or more HMOs is a trisaccharide selected from the group consisting of LNT-2, 3′-SL and 6′-SL, or a tetrasaccharide selected from the group consisting of LNT, LNnT or FSL.
14 . The genetically modified cell of claim 13 , wherein the one or more HMOs is 3′-SL or 6′-SL.
15 . The genetically modified cell of claim 13 , wherein the one or more HMOs comprises LNT, LNnT, or FSL.
16 . A nucleic acid construct comprising a nucleic acid sequence encoding a protein of SEQ ID NO: 1, or a functional homologue thereof, having more than 90% sequence identity to SEQ ID NO: 1, wherein the nucleic acid sequence encoding a protein of SEQ ID NO: 1, has at least 80% sequence identity to SEQ ID NO: 2, wherein the construct further comprises a nucleic acid sequence comprising an expression element comprising a lac promoter or a glp promoter.
17 . The nucleic acid construct according to claim 16 , wherein the lac promoter, if present, is Plac and the glp promoter, if present, is PglpF.
18 . A method for the production of one or more HMOs, the method comprising the steps of:
(i) providing the genetically modified cell of claim 1 ;
(ii) culturing the cell according to (i) in a suitable cell culture medium to express the recombinant nucleic acid, whereby one or more HMOs are produced by the cultured genetically modified cell;
(iii) harvesting the one or more HMOs produced in step (ii).
19 . The method of claim 18 , wherein the one or more HMOs is a trisaccharide selected from the group consisting of LNT-2, 3′-SL and 6′-SL, or a tetrasaccharide selected from the group consisting of LNT, LNnT or FSL.
20 . The method of claim 18 , wherein the one or more HMOs is 3′-SL or 6′-SL.