Metabolic engineering
AssigneePlant Bioscience Limited
InventorsAnne Osbourn, James Reed
Life of this patent
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Abstract
The invention relates generally to materials and methods for biosynthesising quillaic acid in a host by expressing heterologous nucleotide sequences in the host each of which encodes a polypeptide which in combination have said QA biosynthesis activity. Example polypeptides include (i) a Beta-amyrin synthase; (ii) an enzyme capable of oxidising Beta-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid; (iii) an enzyme capable of oxidising Beta-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol; and (iv) an enzyme capable of oxidising Beta-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde. Preferred nucleotide sequences are obtained from, or derived from, Q. saponaria.
Claims24
A method of converting a host from a phenotype whereby the host is unable to carry out quillaic acid (QA) biosynthesis from 2,3-oxidosqualene (OS) to a phenotype whereby the host is able to carry out said QA 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 a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have said QA biosynthesis activity,
- wherein the nucleic acid encodes all of the following polypeptides:
- (i) a β-amyrin synthase (bAS) for cyclisation of OS to a triterpene, wherein the bAS has an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 2;
- (ii) a C-28 oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid, wherein the CYP450 C-28 oxidase has an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 4, SEQ ID NO:
- 18 or a polypeptide the encoded by any of SEQ ID NOs:
- 19-28;
- (iii) a C-16α oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol, wherein the C-16α oxidase has an amino acid sequence that is at least 90% identical to any of SEQ ID NOS:
- 6, 10 or 12;
- and
- (iv) a C-23 oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde, wherein the C-16α oxidase has an amino acid sequence that is at least 90% identical to any of SEQ ID NOS:
- SEQ ID:
- No 8, 14 or 16.
A host cell containing or transformed with a heterologous nucleic acid which comprises a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have quillaic acid (QA) from 2,3-oxidosqualene (OS) biosynthesis activity,
- wherein the heterologous nucleic acid encodes all of the following polypeptides:
- (i) a β-amyrin synthase (bAS) for cyclisation of OS to a triterpene, wherein the bAS has an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 2;
- (ii) a C-28 oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid, wherein the CYP450 C-28 oxidase has an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 4, SEQ ID NO:
- 18 or a polypeptide the encoded by any of SEQ ID NOs:
- 19-28;
- (iii) a C-16α oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol, wherein the C-16α oxidase has an amino acid sequence that is at least 90% identical to any of SEQ ID NOS:
- 6, 10 or 12;
- and
- (iv) a C-23 oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde, wherein the C-16α oxidase has an amino acid sequence that is at least 90% identical to any of SEQ ID NOS:
- SEQ ID:
- No 8, 14 or 16;
- and
- wherein expression of said nucleic acid imparts on the transformed host the ability to carry out QA biosynthesis.
A host cell containing or transformed with nucleic acids encoding:
- (i) a β-amyrin synthase (bAS) for cyclisation of OS to a triterpene, wherein the bAS has an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 2;
- (ii) a C-28 oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid, wherein the CYP450 C-28 oxidase has an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 4, SEQ ID NO:
- 18 or a polypeptide the encoded by any of SEQ ID NOs:
- 19-28;
- (iii) a C-16α oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol, wherein the C-16α oxidase has an amino acid sequence that is at least 90% identical to any of SEQ ID NOS:
- 6, 10 or 12;
- and
- (iv) a C-23 oxidase capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde, wherein the C-16α oxidase has an amino acid sequence that is at least 90% identical to any of SEQ ID NOS:
- SEQ ID:
- No 8, 14 or 16,
- wherein the host cell carries out QA biosynthesis.
Dependencies are inferred from the claim text.