US 12,391,963 B2
Metabolic engineering
Anne Osbourn, Norwich (GB); and James Reed, Norwich (GB)
Assigned to PLANT BIOSCIENCE LIMITED, Norwich (GB)
Appl. No. 16/762,097
Filed by PLANT BIOSCIENCE LIMITED, Norwich (GB)
PCT Filed Dec. 20, 2018, PCT No. PCT/EP2018/086430
§ 371(c)(1), (2) Date May 6, 2020,
PCT Pub. No. WO2019/122259, PCT Pub. Date Jun. 27, 2019.
Claims priority of application No. 1721600 (GB), filed on Dec. 21, 2017.
Prior Publication US 2023/0279444 A1, Sep. 7, 2023
Int. Cl. C12P 5/00 (2006.01); C12N 9/02 (2006.01); C12N 9/04 (2006.01); C12N 9/10 (2006.01); C12N 9/90 (2006.01); C12N 15/81 (2006.01); C12N 15/82 (2006.01)
CPC C12P 5/007 (2013.01) [C12N 9/0006 (2013.01); C12N 9/0042 (2013.01); C12N 9/0073 (2013.01); C12N 9/1085 (2013.01); C12N 9/90 (2013.01); C12N 15/81 (2013.01); C12N 15/8243 (2013.01); C12Y 101/01034 (2013.01); C12Y 101/01088 (2013.01); C12Y 106/02004 (2013.01); C12Y 205/01021 (2013.01); C12Y 504/99039 (2013.01)] 24 Claims
 
1. 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.