Biosynthesis
OwnerAnne OSBOURN, James REED +3inventors
InventorsAnne OSBOURN, James REED, Anastasia ORME, Laetitia MARTIN, Charlotte OWEN
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Abstract
The present invention relates to a biosynthetic route to the QS-21 and QS-18 molecules including the C-18 acyl chain and precursors thereof, as well as enzymes involved, the products produced and uses of the products.
Claims55
A method of making QA*-F*-C18-A, wherein the F* chain is at the C-28 position of QA*, and the C-18-A chain is attached to the D-fucose of the F* chain, the method comprising the step of combining QA*-F*-C18 with an enzyme capable of transferring UDP-β-L-arabinofuranose to QA*-F*-C18 to form QA*-F*-C18-A.
A method of making QA*-F*-C18-A-G wherein the F* chain is at the C-28 position of QA*, the C-18-A chain is attached to the D-fucose of the F* chain and the G residue is attached at the C-3 position of the rhamnose residue of the F* chain, wherein F* is FRX, FRXX, FRXA or mixtures thereof, preferably wherein F* is FRXA, and wherein the method comprises the step of combining QA*-F* with an enzyme capable of transferring a D-glucose residue to QA*-F* to form QA*-F*-G.
A method of making a biosynthetic QA*-F*-C18-A in a host, wherein the F* chain is at the C-28 position of QA*, and the C-18-A chain is attached to the D-fucose of the F* chain, the method comprising the steps of:
- a) expressing genes required for the biosynthesis of QA*-F* into the host, and
- b) introducing a polynucleotide encoding:
- i. at least one or more enzymes capable of transferring a coenzyme A to 2-methylbutyric acid to form 2-methylbutyryl-CoA;
- ii. at least one or more enzymes capable of adding malonyl-CoA, and one or more enzymes capable of reducing a ketone, to form (3S,5S,6S)-3,5-dihydroxy-6-methyloctanoyl-CoA;
- iii. at least one or more enzymes capable of transferring an acyl unit to QA*-F* to form QA*-F*-C9;
- iv. at least one or more enzymes capable of transferring an acyl unit to QA*-F*-C9 to form QA*-F*-C18;
- and
- v. at least one or more enzymes capable of transferring UDP-β-L-arabinofuranose to QA*-F*-C18 to form QA*-F*-C18-A,
- into the host.
A carboxyl CoA enzyme having the amino acid sequence of SEQ ID NO 54 (1CCL), SEQ ID NO 56 (2CCL), SEQ ID NO 58 (3CCL), SEQ ID NO 60 (4CCL), SEQ ID NO 62 (5CCL), SEQ ID NO 64 (6CCL) or an enzyme having an amino acid sequence with at least 60% sequence identity to SEQ ID NO 54, 56, 58, 60, 62 or 64.
A chalcone synthase-like enzyme having the amino acid sequence of SEQ ID NO 66 (ChSA), SEQ ID NO 68 (ChSB), SEQ ID NO 70 (ChSC), SEQ ID NO 72 (ChSD), SEQ ID NO 74 (ChSE), SEQ ID NO 76 (ChSF) or an enzyme having an amino acid sequence with at least 50% sequence identity to SEQ ID NO 66, 68, 70, 72, 74 or 76.
A keto reductase enzyme having the amino acid sequence of SEQ ID NO 78 (KR11) or an enzyme having an amino acid sequence with at least 20% sequence identity to SEQ ID NO 78.
A keto reductase enzyme having the amino acid sequence of SEQ ID NO 80 (KR23′) or an enzyme having an amino acid sequence with at least 15% sequence identity to SEQ ID NO 80.
An acyl transferase enzyme having the amino acid sequence of SEQ ID NO 82 (DMOT9) or an enzyme having an amino acid sequence with at least 25% sequence identity to SEQ ID NO 82.
An acyl transferase enzyme having the amino acid sequence of SEQ ID NO 84 (DMOT4) or an enzyme having an amino acid sequence with at least 15% sequence identity to SEQ ID NO 84.
An arabinofuranosyl transferase enzyme having the amino acid sequence of SEQ ID NO 86 (UGT-L-short), SEQ ID NO 88 (UGT-L-long), or an enzyme having an amino acid sequence with at least 45% sequence identity to SEQ ID NO 86 or 88.
Dependencies are inferred from the claim text.