Production of milk oligosaccharides in plants
View Patent ↗Mammalian milk oligosaccharides (MMO) are produced in plants engineered to express recombinant MMO biosynthetic pathways.
1 . A method of producing a mammalian milk oligosaccharide (MMO) in a plant or plant part, comprising growing a plant comprising and expressing a recombinant MMO biosynthetic pathway sufficient to produce the MMO, wherein the pathway produces the MMO in the plant or plant part, wherein the pathway comprises recombinant enzymes:
a β1-4 galactosyltransferase (β1-4-GalT), which glycosylates glucose using UDP-galactose to make lactose; and
a β1-3 N-acetylglucosaminyltransferase (1-3-GlcNAc), which generates lacto-N-triose (LNTII) through the transfer of GlcNAC from UDP-GlcNAc to the galactose in the lactose; and at least one of:
a β1-3-galactosyltransferase (β1-3-GalT) and a β1-4-galactosyltransferase (β1-4-GalT), which converts the LNTII to lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), respectively,
wherein the plant is tobacco.
2 . A method of claim 1 , wherein the pathway comprises recombinant enzyme:
a α1-2-fucosyltransferase (α1-2-FucT) to produce lacto-N-fucopentaose I (LNFPI), a fucosylated pentasaccharide generated by the addition of an α-1,2-linked fucose to the terminal galactose in the lacto-N-tetraose (LNT).
3 . The method of claim 1 , wherein the pathway comprises recombinant enzyme:
a α2-3-sialyltransferase or a α2-6-sialyltransferase to add an α-2,6-linked or α-2,3-linked Neu5Ac to the lacto-N-neotetraose (LNnT) to produce sialyllacto-N-neotetraose d (LSTd) or sialyllacto-N-neotetraose c (LSTc), respectively.
4 . The method of claim 1 , wherein the plant is comprising and expressing a eukaryotic or prokaryotic nucleotide sugar biosynthetic pathway sufficient to increase the production of the MMO.
5 . The method of claim 1 , wherein the plant is further comprising and expressing a prokaryotic nucleotide sugar biosynthetic pathway sufficient to increase the production of the MMO, wherein the pathway comprises one, two or three of the following pathways and corresponding enzymes:
a) GDP-fucose pathway and enzymes: ManA, ManB, ManC, Gmd, and WcaG,
b) UDP-galactose pathway and enzymes: pgi, pgm, GalU, and GalE, and
c) UDP-GlcNAc pathway and enzymes: glmS, glmM, and glmU.
6 . The method of claim 1 , wherein the MMO is of a mammal selected from human, cow and goat, preferably wherein the MMO is a human milk oligosaccharide (HMO).
7 . The method of claim 1 , the plant comprising and expressing a plurality of recombinant MMO biosynthetic pathways sufficient to produce a plurality of different MMOs.
8 . The method of claim 1 , wherein:
the β1-4 galactosyltransferase (β1-4-GalT) is GalTPM1141;
the β1-3 N-acetylglucosaminyltransferase (β1-3-GnT) is NmLgtA;
the β1-3-galactosyltransferase (β1-3-GalT) is Cvβ3GalT;
the β1-4-galactosyltransferase (β1-4-GalT) is HP0826 or NmLgtB;
the α1-2-fucosyltransferase (α1-2-FucT) is Te2FT;
the α2-3-sialyltransferase is PmSt3); and
the α2-6-sialyltransferase is St6.
9 . The method of claim 1 , wherein the plant or plant part is comprising and expressing of a β1-4-GalT, a β1-3-GlcNAcT, a β1-4-GalT, and a 1-3-GalT to produce LNnT and LNT.
10 . The method of claim 1 , wherein the plant or plant part is producing non-fucosylated neutral MMOs (nMMOs) comprising a lactose core with decorations of galactose and N-acetylglucosamine (GlcNAc).
11 . The method of claim 1 , wherein the plant or plant part is producing fucosylated MMOs, particularly comprising a lactose core decorated with one or more fucose moieties.
12 . The method of claim 1 , wherein the plant or plant part is comprising and expressing a β1-4-GalT, a β1-3-GlcNAcT, a β1-4-GalT, a β1-3-GalT and a α1-2-fucosyltransferase (α1-2-FucT) to produce lacto-N-fucopentaose I (LNFPI), a fucosylated pentasaccharide generated by the addition of an α-1,2-linked fucose to the terminal galactose in lacto-N-tetraose (LNT).
13 . The method of claim 1 , wherein the plant or plant part is producing fucosyllactose, lacto-N-tetraose or lacto-N-neotetraose, and lacto-N-fucopentaose I.
14 . The method of claim 1 , wherein to improve LNFPI yields, nucleotide sugar biosynthetic pathways ( FIG. 1 B ) are expressed to increase the availability of GDP-fucose, UDP-galactose, and UDP-GlcNAc, combinatorially.
15 . The method of claim 1 , wherein the pathway comprises recombinant enzyme:
a α1-2-fucosyltransferase (α1-2-FucT) to produce lacto-N-fucopentaose I (LNFPI), a fucosylated pentasaccharide generated by the addition of an α-1,2-linked fucose to the terminal galactose in the lacto-N-tetraose (LNT), and
the plant further comprises a prokaryotic GDP-fucose biosynthetic pathway comprising enzymes: ManA, ManB, ManC, Gmd and WcaG.
16 . The method of claim 1 , producing CMP-Neu5Ac.
17 . The method of claim 1 , wherein the plant or plant part is comprising and expressing a three gene bacterial pathway comprising genes: neuA, neuB, and neuC, or a four gene mammalian pathway comprising genes: GNE, NANS, NANP, and CMAS, which utilize UDP-GlcNAc as a precursor for the production of CMP-Neu5Ac.
18 . The method of claim 1 , wherein the plant or plant part is comprising and expressing a mammalian CMP-Neu5Ac pathway, comprising genes: GalTPM1141, NmLgtA, Cvβ3GalT, and Hp0826 for NmLgtB, and either PmSt3 or St6, to produce 3′SL, 6′SL and isomers of LST.
19 . The method of claim 1 , wherein the plant is a stable, transgenic plant, comprising constructs for the constitutive production of 2′FL and LNFPI.
20 . The method of claim 1 , further comprising isolating the MMO from the plant or plant part.