| CPC C12N 15/8243 (2013.01) [C12P 5/007 (2013.01); C12Y 204/01017 (2013.01)] | 33 Claims |
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1. A method of converting a host from a phenotype whereby the host is unable to perform the biosynthesis of the 3-O branched trisaccharide quillaic acid (“QA”) derivative (“QA-3-O-TriS”), which QA-3-O-TriS is
3β-{[β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid]oxy}-quillaic acid (QA-GlcpA-[Galp]-Xylp) or
(3β-{[α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid]oxy}-quillaic acid) (QA-GlcpA-[Galp]-Rhap),
by glycosylation of the 3-O position of QA, to a phenotype whereby the host is able to carry out said QA-3-O-TriS biosynthesis,
which method comprises the step of expressing a heterologous nucleic acid within the host or one or more cells thereof, following an earlier step of introducing the nucleic acid into the host or an ancestor of either,
wherein the heterologous nucleic acid comprises nucleotide sequences each of which encodes a polypeptide which in combination have said QA-3-O-TriS biosynthesis activity,
wherein the heterologous nucleic acid encodes two or three of the following types of polypeptide (i), (ii) or (iii):
(i) a QA 3-O glucuronosyl transferase (“QA-GlcAT”) capable of transferring D-glucuronic acid (“GlcpA”) at the 3-O position of quillaic acid to form 3β-{[β-d-glucopyranosiduronic acid]oxy}-quillaic acid (“QA-GlcpA”);
(ii) a QA-GlcpA galactosyl transferase (“QA-GalT”) capable of transferring D-Galactose (“Galp”) via a β-1->2 linkage to QA-GlcpA to form 3β-{[β-d-galactopyranosyl-(1->2)-β-d-glucopyranosiduronic acid]oxy}-quillaic acid (“QA-GlcpA-Galp”);
(iii) a QA-GlcpA-Galp Rhamnosyl/Xylosyl transferase (“QA-RhaT/XylT”), capable of transferring 1-Rhamnose (“Rhap”) and/or d-Xylose (“Xylp”) via a 1,3 linkage to QA-GlcpA-Galp to form (3β-{[α-l -rhamnopyranosyl-(1->3)-[β-d-galactopyranosyl-(1->2)]-β-d-glucopyranosiduronic acid]oxy}-quillaic acid) (“QA-GlcpA-[Galp]-Rhap”) and/or 3β-{[β-d-xylopyranosyl-(1->3)-[β-d-galactopyranosyl-(1->2)]-β-d-glucopyranosiduronic acid]oxy}-quillaic acid (“QA-GlcpA-[Galp]-Xylp”) respectively;
wherein:
the QA-GlcAT of (i) comprises the amino acid sequence of SEQ ID: No 2 or 26, or an amino acid sequence that is at least 80% identical to SEQ ID: No 2 or 26;
the QA-GalT of (ii) comprises the amino acid sequence of SEQ ID: No 4, or an amino acid sequence that is at least 80% identical to SEQ ID: No 4; and
the QA-RhaT/XylT of (iii) comprises the amino acid sequence of SEQ ID: No 6, 28, 30, or 32, or an amino acid sequence that is at least 80% identical to SEQ ID: No 6, 28, 30, or 32 therewith.
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