Claims

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 a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have said QA-3-O- TriS biosynthesis activity.

2 A method as claimed in claim 1, 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 L-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 each of the polypeptides is optionally from Quillaja saponaria.

***

3 A method as claimed in claim 2, wherein the heterologous nucleic acid encodes all three types of polypeptide.

4 A method as claimed in claim 2 or claim 3, wherein the nucleotide sequences are from Q. saponaria.

5 A method as claimed in claim 3, wherein

(i) the polypeptides are selected from the QA-GlcAT, QA-GalT, and QA-RhaT/XylT enzymes in Table 5 or 6, or from substantially homologous variants or fragments of any of said polypeptides in Tables 5 or 6, and/or

(ii) the nucleotide sequences are selected from the QA-GlcAT, QA-GalT, and QA- RhaT/XylT nucleotide sequence in Table 5 or 6 or from substantially homologous variants or fragments of any of said nucleotides sequences in Tables 5 or 6.

6 A method as claimed in claim 4, wherein the respective polypeptides are selected from the lists consisting of:

(i) the QA-GlcAT shown in SEQ ID: No 2 or 26;

(ii) the QA-GalT shown in SEQ ID: No 4;

(iii) a QA-RhaT/XylT shown in SEQ ID: No 6 28, 30, or 32;

or substantially homologous variants or fragments of any of said respective polypeptides.

***

7 A method as claimed in any one of claims 1 to 6, wherein the heterologous nucleic acid further comprises a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have QA biosynthesis activity (“QA polypeptide”), wherein the nucleic acid encodes all of the following QA polypeptides:

(i) a β-amyrin synthase (bAS) for cyclisation of 2,3-oxidosqualene (OS) to a triterpene;

(ii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid (“C-28 oxidase”);

(iii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C- 16α position to an alcohol (“C-16α oxidase”); and

(iv) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde (“C-23 oxidase”),

wherein each of the polypeptides is optionally from Q. saponaria.

8 A method as claimed in claim 7, wherein the C-28 oxidase, C-16α oxidase, and C- 23 oxidase are all CYP450 enzymes.

9 A method as claimed in claim 8, wherein

(i) the C-28 oxidase is a CYP716;

(ii) the C-16α oxidase is a CYP716 or CYP87;

(iii) the C-23 oxidase is a CYP714, CYP72, or CYP94.

10 A method as claimed in any one of claims 7 to 9, wherein the QA polypeptides are selected from the list consisting of: the β-amyrin synthase (bAS) shown in SEQ ID: No 12; the C-28 oxidase shown in SEQ ID: No 14;

the C-16α oxidase shown in SEQ ID: No 16;

the C-23 oxidase shown in the SEQ ID: No 18;

or substantially homologous variants or fragments of any of said polypeptides.

****

11 A method as claimed in any one of claims 1 to 10, wherein the nucleic acid further comprises a plurality of nucleotide sequences encoding one or more of the following polypeptides:

(i) an HMG-CoA reductase (HMGR);

(ii) a squalene synthase (SQS)

wherein the HMGR or SQS are optionally selected from the respective polypeptides in Table 7 or substantially homologous variants or fragments of any of said polypeptides, or are encoded by the respective polynucleotides in Table 7, or substantially homologous variants or fragments of any of said polynucleotides.

***

12 A method as claimed in any one of claims 1 to 11, wherein the nucleotide sequences are present on two or more different nucleic acid molecules.

13 A method as claimed in claim 12, wherein the host is a plant and the nucleic acid molecules are introduced by co-infiltration with a plurality of Agrobacterium tumefaciens strains each carrying one or more of the nucleic acid molecules.

14 A method as claimed in claim 13, wherein the nucleic acid molecules are transient expression vectors.

15 A method as claimed in claim 14, wherein each of the transient expression vectors comprises an expression cassette comprising:

(i) a promoter, operably linked to

(ii) an enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated;

(iii) a nucleotide sequence encoding one of the polypeptides which in combination have said QA-3-O-TriS biosynthesis activity;

(iv) a terminator sequence; and optionally

(v) a 3 UTR located upstream of said terminator sequence.

***

16 A method as claimed in any one of claims 1 to 15, wherein the host is a plant which is converted such as to have a modified QA-3-O-TriS content.

***

17 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 QA-3-O-TriS biosynthesis activity,

wherein expression of said nucleic acid imparts on the transformed host the ability to carry out QA-3-O-TriS biosynthesis,

wherein the host cell is optionally obtainable by the method of any one of claims 1 to 16.

18 A host cell as claimed in claim 17 containing or transformed with a heterologous nucleic acid which further comprises a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have QA biosynthesis activity as defined in any of claims 7 to 10.

***

19 A process for producing the host cell of claim 17 or claim 18 by co-infiltrating a plurality of recombinant constructs comprising said nucleic acid into the cell for transient expression thereof.

20 A process for producing the host cell of claim 17 or claim 18 by transforming a cell with heterologous nucleic acid by introducing said nucleic acid into the cell via a vector and causing or allowing recombination between the vector and the cell genome to introduce the nucleic acid into the genome.

21 A method for producing a transgenic plant, which method comprises the steps of:

(a) performing a process as claimed in claim 20 wherein the host cell is a plant cell, and

(b) regenerating a plant from the transformed plant cell.

***

22 A transgenic plant which is obtainable by the method of claim 21, or which is a clone, or selfed or hybrid progeny or other descendant of said transgenic plant, wherein expression of said heterologous nucleic acid imparts an increased ability to carry out QA-3-O-TriS synthesis compared to a wild-type plant otherwise corresponding to said transgenic plant.

23 A transgenic plant comprising nucleic acid encoding 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 L-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 at least one said nucleic acids is heterologous nucleic acid,

and optionally comprising nucleic acid encoding the following types of polypeptide:

(iv) a β-amyrin synthase for cyclisation of 2,3-oxidosqualene to a triterpene;

(v) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid;

(vi) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol; and

(vii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde,

wherein at least one said nucleic acids is heterologous nucleic acid,

and wherein optionally each of the polypeptides is from Quillaja saponaria,

24 An in vivo or in vitro method of transferring D-glucuronic acid at the 3-O position of quillaic acid to form 3β-{[β-D-glucopyranosiduronic acid]oxy}-quillaic acid, the method comprising contacting the quillaic acid with a cellulose synthase enzyme, which is optionally selected from SEQ ID NO: 2, SEQ ID NO: 26, or homologous variant of either.

***

25 An isolated nucleic acid molecule which nucleic acid comprises a QA-3-O-TriS-biosynthetic nucleotide sequence which:

(i) encodes all or part of polypeptide SEQ ID NO: 2, 4, 6, 26, 28, 30 or 32 or

(ii) encodes a variant polypeptide which is a homologous variant of any of these SEQ ID Nos which shares at least about 60% identity with said SEQ ID NOs, which variant polypeptide in each case has the respective activity of said SEQ ID NO shown in Table 5.

26 A nucleic acid as claimed in claim 25 wherein the nucleotide sequence is selected from SEQ ID NO: 1, 3, 5, 25, 27, 29, or 31 or the genomic equivalent thereof.

27 A nucleic acid as claimed in claim 25 wherein the nucleotide sequence consists of an allelic or other homologous or orthologous variant of the nucleotide sequence of claim 26.

28 A nucleic acid as claimed in claim 25 wherein the nucleotide sequence is derived or obtained from Q. saponaria.

***

29 A nucleic acid as claimed in claim 28 wherein the QA-3-O-TriS-biosynthetic nucleotide sequence encodes a derivative of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 26, 28, 30 or 32 by way of addition, insertion, deletion or substitution of one or more amino acids.

***

30 A process for producing a nucleic acid as claimed in claim 29 comprising the step of modifying a nucleic acid as claimed in any one of claims 26 to 28.

31 A method for identifying or cloning a nucleic acid as claimed in claim 27 or claim 28, which method employs all or part of a nucleic acid as claimed in claim 26 or the complement thereof.

32 A method as claimed in claim 31, which method comprises the steps of:

(a) providing a preparation of nucleic acid from a plant cell;

(b) providing a nucleic acid molecule which is a probe, said nucleic acid molecule having a sequence, which sequence is present in a nucleotide sequence of claim 26, or in a complementary sequence thereof;

(c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation; and,

(d) identifying nucleic acid in said preparation which hybridises with said nucleic acid molecule.

33 A method as claimed in claim 32, which method comprises the steps of:

(a) providing a preparation of nucleic acid from a plant cell;

(b) providing a pair of nucleic acid molecule primers suitable for PCR, at least one of said primers being a sequence of at least about 16-24 nucleotides in length, which sequence is present in a nucleotide sequence of claim 26, or in a complementary sequence thereof;

(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR; and,

(d) performing PCR and determining the presence or absence of an amplified PCR product.

34 A method for identifying a nucleic acid as claimed in claim 27 or claim 28, which method employs all or part of the nucleotide sequences as claimed in claim 26 or a complementary sequence thereof as query sequence to interrogate a database of plant

genomic sequences, and identifying the target nucleic acid as claimed in claim 27 or claim 28 based on sequence similarity and clustering or co-expression of the target nucleic acid with the QA-3-O-TriSI-biosynthetic nucleotide sequences.

***

35 A recombinant vector which comprises the nucleic acid of any one of claims 25 to 29.

36 A vector as claimed in claim 35 wherein the nucleic acid is operably linked to a promoter for transcription in a host cell, wherein the promoter is optionally an inducible promoter.

37 A vector as claimed in claim 35 or claim 36 which is a plant vector or a microbial vector.

38 A vector as claimed in claim 37 wherein the vector comprises an expression cassette comprising:

(i) a promoter, operably linked to

(ii) an enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated;

(iii) the nucleic acid of any one of claims 25 to 29;

(iv) a terminator sequence; and optionally

(v) a 3’ UTR located upstream of said terminator sequence.

***

39 A method which comprises the step of introducing one or more different vectors of any one of claims 35 to 38 into a host cell, and optionally causing or allowing recombination between the vector and the host cell genome such as to transform the host cell.

40 The method as claimed in claim 39 wherein the one or more different vectors collectively encode:

(i) the QA-GlcAT shown in SEQ ID: No 2 or 26, or a substantially homologous variant or fragment thereof;

(ii) the QA-GalT shown in SEQ ID: No 4 or a substantially homologous variant or fragment thereof;

(iii) the QA-RhaT/XylT shown in SEQ ID: No 6, 28, 30, or 32, or a substantially homologous variant or fragment thereof;

wherein in each case the substantially homologous variant or fragment of any of said polypeptides has the respective activity of said SEQ ID NO. shown in Table 5

41 A host cell containing or transformed with a vector according to any one of claims 35 to 38.

42 A host cell as claimed in claim 41 which is microbial, optionally a yeast cell.

43 A host cell as claimed in claim 42 which further contains or is transformed with heterologous nucleic acid which comprises one or more nucleotide sequences each of which encodes a polypeptide which is a plant cytochrome P450 reductases (CPR), which is optionally the CPR shown in SEQ ID No: 23 or is a substantially homologous variant or fragment of said polypeptide.

44 A host cell which is a plant cell having a heterologous nucleic acid as claimed in any one of claims 25 to 29 within its chromosome.

45 A host cell as claimed in claim 44 which is a plant cell having a heterologous nucleic acid which is:

(i) the QA-GlcAT shown in SEQ ID: No 2 or 26, or a substantially homologous variant or fragment thereof; and/or

(ii) the QA-GalT shown in SEQ ID: No 4 or a substantially homologous variant or fragment thereof; and/or

(iii) the QA-RhaT/XylT shown in SEQ ID: No 6, 28, 30, or 32, or a substantially homologous variant or fragment thereof; and/or

wherein in each case the substantially homologous variant or fragment of any of said polypeptides has the respective activity of said SEQ ID NO. shown in Table 5

***

46 A method for producing a transgenic plant, which method comprises the steps of:

(a) performing a method as claimed in claim 39 wherein the host cell is a plant cell, and

(b) regenerating a plant from the transformed plant cell.

47 A transgenic plant which comprises a host cell as claimed in any one of claims 41 to 45, or is obtainable by the method of claim 46, or which is a clone, or selfed or hybrid progeny or other descendant of said transgenic plant, which in each case includes a heterologous nucleic acid of any one of claims 25 to 29.

***

48 A plant having a heterologous nucleic acid as claimed in any one of claims 25 to 29 or vector of any one of claims 35 to 38 in one or more of its cells.

***

49 A leaf, stem, or edible portion or propagule from a plant as claimed in claim 22,

23, 47 or claim 48, which in each case includes a heterologous nucleic acid of any one of claims 25 to 28.

50 A host cell of any one of claims 41 to 45 or a plant of claim 36 or claim 47 or claim 48 or a leaf, stem, or edible portion or propagule of claim 49 which further comprises a heterologous nucleic acid comprising all of:

(i) the β-amyrin synthase shown in SEQ ID: No 11;

(ii) the C-28 oxidase shown in SEQ ID: No 13;

(iii) the C-16α oxidase shown in SEQ ID: No 15;

(iv) the C-23 oxidase shown in the SEQ ID: No 16;

or substantially homologous variants or fragments of any of said polynucleotides.

***

51 An isolated, and optionally recombinant, polypeptide which is encoded by the QA- 3-O-TriS-biosynthetic nucleotide sequence of any one of claims 25 to 28.

52 Use of a polypeptide of claim 51 to catalyse QA-glycosylation, optionally according to the biological activity shown in Table 5.

53 Use of a polypeptide of claim 51 in a method of synthesising QA-3-O-TriS.

***

54 A method of making the polypeptide of claim 51, which method comprises the step of expression the polypeptide from a nucleic acid of any one of claims 25 to 28 in a host cell.

55 A method for influencing or affecting the QA-glycosylation in a host, the method comprising the step of:

(i) causing or allowing expression of a heterologous nucleic acid as claimed in any one of claims 25 to 28 within the cells of the host, following an earlier step of introducing the nucleic acid into a cell of the host or an ancestor thereof, or

(ii) introducing a silencing agent capable of silencing expression of a nucleotide sequence as described in claim 26 or claim 27 into a cell of the host or an ancestor thereof.

***

56 A method of producing a product which is a glycosylated QA, which is optionally QA-3-O-TriS, or downstream product thereof, in a host, which is optionally a plant, which method comprises performing a method as claimed in any one of claims 1 to 16, 24, 39, 40, or 55, and optionally isolating the product from the host.

57 A method of producing a product which is a glycosylated QA, which is optionally QA-3-O-TriS, or downstream product thereof in a heterologous host, which method comprises culturing a host cell as claimed in any one of claims 17 to 18, 41 to 45, or claim 50, and purifying the product therefrom.

58 A method of producing a product which is a glycosylated QA, which is optionally QA-3-O-TriS, or a derivative thereof in a heterologous plant, which method comprises growing a plant as claimed in any one of claims claim 23, 47 or claim 48 and then harvesting the a glycosylated QA and purifying the product therefrom.

59 Use of a glycosylated QA, which is optionally QA-3-O-TriS, or a derivative thereof, obtained by the method of any one of claims 55 to 58 in the preparation of an adjuvant.

60 A method, plant, vector, host cell, leaf, stem, or edible portion or propagule, as claimed in any one of claims 16, 22, 23, 32-34, 37, 43-50, 56-58 which is a crop plant or a moss.

***

61 Double-stranded RNA which comprises an RNA sequence equivalent to part of a nucleotide sequence as described in claim 26, which is optionally a siRNA duplex consisting of between 20 and 25 bps.

***

62 An antibody which specifically binds the polypeptide of claim 51.

Transferase enzymes

Technical field

The present invention relates generally to genes and polypeptides which have utility in modifying quillaic acid in host cells. The invention further relates to systems, methods and products employing the same.

Background to the invention

Plants produce a wide variety of cyclic triterpenes, such as sterols and triterpenoids, which are the major products of the mevalonate pathway.

QS-21 is a complex triterpenoid saponin synthesised by the Chilean tree Quillaja saponaria (order Fabales) (Figure 1). QS-21 is a potent immunostimulatory agent capable of enhancing antibody responses and boosting specific T-cell responses, giving it significant adjuvant potential [1, 2]. The AS01 adjuvant is a liposomal formulation of QS-21 and 3-O-desacyl-4'-monophosphoryl lipid A and is currently licenced as part of the GlaxoSmithKline ‘Shingrix’ vaccine against herpes zoster and ‘Mosquirix’ against malaria [1, 3]. As these represent the first licenced human vaccines to utilise QS-21, demand for this molecule is expected to increase substantially over the coming years.

Currently, QS-21 is naturally sourced. Engineering production of QS-21 (or a suitable intermediate for semi-synthesis) in a heterologous host would provide an alternative or addition to current production methods. Presently however, little is known about the biosynthesis of this QS-21 which provides a significant barrier to realising this objective.

Biochemically, QS-21 consists of a C-30 triterpenoid backbone known as quillaic acid. This scaffold is decorated with a branched trisaccharide at the C-3 position and a linear tetrasaccharide at the C-28 position. The terminal sugar of the tetrasaccharide may be either β-D-apiose or β-D-xylose. Finally, the β-D-fucose sugar within the tetrasaccharide also features a C-18 acyl chain which is glycosylated with an arabinose sugar (Figure 1).

Biosynthesis of quillaic acid proceeds from β-Amyrin which is synthesised through cyclisation of the universal linear precursor 2,3-oxidosqualene (OS) by oxidosqualene cyclases (OSCs) (Figure 2). The β-amyrin scaffold is further oxidised with a carboxylic acid, alcohol and aldehyde at the C-28, C-16α and C-23 positions, respectively, to form quillaic acid (QA). A proposed biosynthetic pathway for this is given in Figure 2.

Prior-filed unpublished PCT/EP2018/086430 (subsequently published as WO 2019/122259) describes the identification of enzymes participating in QA production. Candidate enzymes were cloned from leaf cDNA. Enzymes were tested by transient co-expression in Nicotiana benthamiana, allowing for the identification of one OSC and three cytochrome P450s (P450s) required for synthesis of β-amyrin and oxidation to quillaic acid (Figure 2). These enzymes may be referred to herein as QsbAS (β-amyrin synthase) and QsCYP716-C-28, QsCYP716-C-16α and QsCYP714-C-23 oxidases.

A proposed scheme for the glycosylation of quillaic acid to 3β-{[β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid]oxy}-quillaic acid (QA-GlcpA-[Galp]-Xylp) is shown in Figure 3.

Some enzymes reported to be capable of glycosylating triterpenes have previously been obtained from plants other than Q. saponaria, including a C-3 glucuronic acid (GlcA) transferase from licorice [ Xu, G., et al., A novel glucuronosyltransferase has an unprecedented ability to catalyse continuous two-step glucuronosylation of glycyrrhetinic acid to yield glycyrrhizin. New Phytologist, 2016. 212(1): p. 123-135.] and a C-3-GlcA galactosyltransferase from soybean [ Shibuya, M., et al., Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max. FEBS Lett, 2010. 584(11): p. 2258-64].

US20190059314 and WO/2020/049572 relate to genes reported to be useful in the biosynthesis of steroidal alkaloids and saponins, including regulatory genes and enzyme-encoding genes, and the use thereof for altering the content of steroidal (glyco)alkaloids or phytosterols in plants. US20190059314 discusses the use of a gene encoding a cellulose synthase like protein in this context.

However, glycosyltransferases (GTs) responsible for glycosylation or successive glycosylation at the C-3 position of QA have not been previously reported. In the light of the above it can be seen that the provision of such GTs would provide a contribution to the art.

Disclosure of the invention

The present invention concerns the identification of the glycosyltransferases (GTs) responsible for successive glycosylation at the C-3 position of QA.

As depicted in Figure 3, the branched trisaccharide chain at the C-3 position in QS-21 is initiated with a β-D-glucuronic acid (GlcpA) residue attached at the 3-O position of QA. The GlcpA residue is then linked to a D-Galactose (Galp) via a β-1->2 linkage and to a D-Xylose (Xylp) via a β-1,3 linkage.

Note that these latter two steps (Galp and Xylp linkage to GlcpA) are shown in Figure 3 in a linear fashion for simplicity, but may occur in either order.

Additionally, as explained below, the β- D-Xylp residue may be replaced in certain circumstances with an α-L-Rhamnose residue.

Specifically, the inventors have characterised from Quillaja saponaria two glucuronosyl transferases, a galactosyl transferase, and Rhamnosyl, Xylosyl, and dual Rhamnosyl/Xylosyl transferases which permit glycosylation of the 3-O position of QA with the respective saccharide within the 3-O branched tri saccharide. Accordingly, in the sense of the present invention, in the term “3-O branched trisaccharide”, the number “3” is to be understood as the position C-3 of QA (as depicted in Figure 2).

They have termed these enzymes “QsCSL1” and “QsCSLG2” (glucuronosyl transferases), “Qs-3-O-GalT” (galactosyl transferase), “Qs_0283850”, “DN20529_c0_g2_i8”, “Qs_0283870” and “Qs-3-ORhaT/XylT” (rhamnosyl and/or xylosyl transferases), as provided in Table 5.

Surprisingly, the glucuronosyl transferase enzymes are “cellulose synthase- 1 ike” enzymes. Cellulose synthases are generally associated with cell wall biosynthesis, and the functions of most members are unknown [13].

The present disclosure thus provides new uses (in vivo or in vitro) for such enzymes in QA-glycosylation. The terms “QA-glycosylation” and the like as used herein are used broadly to include also successive glycosylations of QA which has already been glycosylated.

***

The galactosyl transferase, and the Rhamnosyl/Xylosyl transferase enzymes are Family 1 UDP-dependent glycosyltransferases (UGTs). This class of enzymes has been previously identified as being involved in plant specialised glycosylation, although other enzyme classes have been found to play a role in plant specialised metabolite biosynthesis [8-12].

The operation of the enzymes in concert is shown in Figure 9 (again depicted in linear fashion for simplicity).

The present invention thus provides for the engineering into host cells or organisms (for example plants or microorganisms) of the ability to perform the biosynthesis of glycosylated QA, for example the 3-O branched trisaccharide quillaic acid (QA) derivative (“QA-3-O-TriS”). Alternatively, the enzymes may be used in vitro to perform the respective activities reported in Table 5.

They may advantageously be used in combination with enzymes permitting the synthesis of QA from OS, such as are characterised in Prior-filed unpublished PCT/E P2018/086430 (subsequently published as WO 2019/122259), the entire disclosure of which is specifically incorporated herein by cross-reference for this purpose. Examples of such enzymes (defined as “QA polypeptides”) are shown in Table 8.

***

Thus, in one aspect of the invention, there is provided 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-ί-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 a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have said QA-3-O-TriS biosynthesis activity.

By way of example, using the illustrative scheme of Figure 3, the encoded polypeptides (enzymes) could comprise any one or more of the following:

(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 L-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]-Xyip”)

respectively.

In one embodiment, the QA-GlcpA-Galp Rhamnosyl/Xylosyl transferase may be a dual QA-GlcpA-Galp Rhamnosyl/Xylosyl transferase.

Each of the polypeptide or nucleotide sequences is optionally obtained or derived from Q. saponaria.

By way of non-limiting example, the QA-GlcAT, QA-GalT, and QA-RhaT/XylT are selected from the respective enzymes in Table 5 or 6, or substantially homologous variants or fragments of any of said polypeptides in Tables 5 or 6.

The nucleic acid may include polynucleotide sequences encoding the respective enzymes in Table 5 or 6, or substantially homologous variants or fragments of any of said polynucleotides.

Regarding the QA-RhaT/XylT enzymes in Table 5, as explained in the Examples below, Qs-3-O-RhaT/XylT is believed to be a dual Rhamnosyl/Xylosyl transferase.

Both Qs_0283850 and DN20529_c0_g2_i8 are believed to be primarily Rhamnosyl transferases, while Qs_0283870 is believed to be primarily a Xylosyl transferase. It will be appreciated, however, that some level of cross-activity may be present. For brevity, all these types of enzymes may be referred to herein as being Qs-3-O-RhaT/XylT enzymes or having QA-RhaT/XylT activity.

Preferably, all three types of enzymes (QA-GlcAT, QA-GalT, and QA-RhaT/XylT) are provided as part of the method of the invention.

Preferred genes or polypeptides for use in the practice of the invention in relation to QA-GlcAT, QA-GalT, QA-RhaT/XylT activities are shown in Table 5 or 6 herein (and the Sequence Annex) or are substantially homologous variants or fragments of these sequences, having or retaining or encoding the requisite biological activity.

For example, QA-GalT activity may be provided by Qs-3-O-GalT, or alternatively by GmUGT73P2, which has been demonstrated herein to share that activity, or by substantially homologous variants or fragments of either of these sequences, retaining that biological activity.

In preferred embodiments, the one, two, or three polypeptides (i), (ii) or (iii) are selected from the respective amino acid sequences listed in Table 5.

In one embodiment, the respective polypeptides are selected from the list consisting of:

(i) the QA-GlcAT shown in SEQ ID: No 2 or 26, most preferably 2;

(ii) the QA-GalT shown in SEQ ID: No 4;

(iii) the QA-RhaT/XylT shown in SEQ ID: No 6, 28, 30, or 32, most preferably 6;

or substantially homologous variants or fragments of any of said polypeptides.

For brevity, in the context of the present invention, and in particular the methods and uses described herein, the polypeptide or nucleotide sequences of any of Tables 5 and 6 may be referred to herein as “QA-3-O-TriS biosynthetic sequences” or “QA-3-O-TriS sequences” e.g. QA-3-O-TriS genes and QA-3-O-TriS polypeptides.

Variants

In addition to the use of these QA-3-O-TriS genes (and polypeptides), the invention encompasses use of variants of these genes (and polypeptides).

A “variant” QA-3-O-TriS nucleic acid or QA-3-O-TriS polypeptide molecule shares homology with, or is identical to, all or part of the QA-3-O-TriS genes or polypeptides sequences discussed herein.

A variant polypeptide shares the relevant biological activity of the native QA-3-O-TriS polypeptide. A variant nucleic acid encodes the relevant variant polypeptide.

In this context, the “biological activity” of the QA-3-O-TriS polypeptide is the ability to catalyse the respective reaction shown in Fig. 3 and described above and/or the activity set out in the respective Table e.g. Table 5 or 6. The relevant biological activities may be assayed based on the reactions shown in Table 5 in vitro. Alternatively, they can be assayed by activity in vivo as described in the Examples i.e. by introduction of a plurality of heterologous constructs to generate the respective product into a host, which can be assayed by LC-MS or the like.

Variants of Qs-3-O-RhaT/XylT may include those preferentially catalysing D-Xylose (Xylp) or L-Rhamnose (Rhap).

Alignments for the purpose of assessing homology may be obtained e.g. using Clustal Omega (version 1.2.4 - accessed through https://www.ebi.ac.uk; see e.g. Sievers F, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7 (1):539.

Variants of the sequences disclosed herein preferably share at least 50%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, or 70%, or 80% identity, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identity with a reference polypeptide or polynucleotide sequence. Such variants may be referred to herein as “substantially homologous”. Preferred variants share at least 80% identity.

Preferred variants may be:

(i) Naturally occurring nucleic acids such as alleles (which will include polymorphisms or mutations at one or more bases) or pseudoalleles (which may occur at closely linked loci to the QA-3-O-TriS genes of the invention). Also included are paralogues, isogenes, or other homologous genes belonging to the same families as the QA-3-O-TriS genes of the invention, for example sharing clades or sub-clades. Also included are orthologues or homologues from other plant species.

Homology may be at the nucleotide sequence and/or amino acid sequence level, as discussed below.

(ii) Artificial nucleic acids, which can be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the sequence of a QA-3-O-TriS gene of the invention.

Also included are nucleic acids corresponding to those above, but which have been extended at the 3' or 5' terminus.

The term “QA-3-O-TriS variant nucleic acid” as used herein encompasses all of these possibilities. When used in the context of polypeptides or proteins it indicates the encoded expression product of the variant nucleic acid.

In each case, the preferred QA-3-O-TriS -biosynthesis modifying nucleic acids are any of SEQ ID Nos 1, 3, 5, or 25, 27, 29 or 31, or substantially homologous variants thereof. In one embodiment (of this, or other aspects of the invention concerning these sequences) they are any of SEQ ID Nos 1, 3, 5, or substantially homologous variants thereof.

The preferred QA-3-O-TriS-biosynthesis modifying polypeptides are any of SEQ ID Nos 2, 4, 6, 26, 28, 30 or 32, or substantially homologous variants thereof. In one embodiment (of

this, or other aspects of the invention concerning these sequences), they are any of SEQ ID Nos 2, 4, 6, or substantially homologous variants thereof.

Other preferred QA-3-O-TriS-biosynthesis modifying nucleic acids for use in the invention are SEQ ID No 19, or substantially homologous variants or fragments thereof. Other preferred QA-3-O-TriS-biosynthesis modifying polypeptides are polypeptides encoded by any of these sequences or variants or fragments e.g. SEQ ID No 20.

Supplementary genes

In embodiments of the invention, in addition to the QA-3-O-TriS genes and variant nucleic acids of the invention described herein, it may be preferable to introduce additional genes which may affect flux of QA-3-O-TriS production.

As explained in prior-filed unpublished PCT /E P2018/086430 (subsequently published as WO 2019/122259), the core aglycone of QS-21 (i.e. QA) is a derivative of the simple triterpene, β-amyrin, which is in turn synthesised by cyclisation of the universal linear precursor 2,3-oxidosqualene (OS) by oxidosqualene cyclases (OSCs).

The β-amyrin scaffold is further oxidised with an alcohol, aldehyde and carboxylic acid at the C-16α, C-23 and C-28 positions, respectively, to form quillaic acid.

QA biosynthesis from OS thus includes at least four different enzymatic steps. The enzymes involved include:

• an oxidosqualene cyclase;

• an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C- 28 position to a carboxylic acid;

• an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof such as oleanolic acid at the C-16α position to an alcohol;

• an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof such as echinocystic acid at the C-23 position to an aldehyde.

For example:

(i) a β-amyrin synthase (bAS) for cyclisation of 2,3-oxidosqualene (OS) to a triterpene;

(ii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid (“C-28 oxidase”);

(iii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol (“C-16α oxidase”); and

(iv) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde (“C-23 oxidase”),

For brevity, these enzymes may be referred to as “bAS”, “C-28 oxidase”, “C-16α oxidase”, and “C-23 oxidase” respectively herein.

For further brevity, these enzymes may be referred to collectively as “QA polypeptides”

herein.

The present invention may be advantageously applied in conjunction with these QA polypeptides and encoding-nucleic acids.

Thus it will be appreciated that the use of the aforementioned QA polypeptides and genes in conjunction with the QA-3-O-TriS polypeptides and genes is expressly envisaged in relation to the any aspects of the invention relating to the materials or methods of QA-3-O-TriS biosynthesis.

In one embodiment:

The C-28 oxidase is a CYP716

The C-16α is a CYP716 or CYP87

The C-23 oxidase is a CYP714, CYP72 or CYP94

Preferred QA genes or QA polypeptides are shown in the Table 8 or biologically active fragments or variants of these. Variants may be homologues, alleles, or artificial derivatives etc. as discussed in relation to QA-3-O-TriS genes or polypeptides as described above.

For example, the QA polypeptides may be any one or more (preferably all) of:

(i) the β-amyrin synthase (bAS) shown in SEQ ID: No 12;

(ii) the C-28 oxidase shown in SEQ ID: No 14;

(iii) the C-16α oxidase shown in SEQ ID: No 16;

(iv) the C-23 oxidase shown in the SEQ ID: No 18;

or substantially homologous variants or fragments of any of said polypeptides.

Mevalonic acid (MVA) is an important intermediate in triterpenoid synthesis. Therefore, it may be desirable to express rate-limiting MVA pathway genes into the host, to maximise yields of QA.

HMG-CoA reductase (HMGR) is believed to be a rate-limiting enzyme in the MVA pathway.

The use of a recombinant feedback-insensitive truncated form of HMGR (tHMGR) has been demonstrated to increase triterpene (b-amyrin) content upon transient expression in N. benthamiana [19].

Thus one embodiment of the invention comprises the use of a heterologous HMGR (e.g. a feedback-insensitive HMGR) along with the QA-3-O-TriS genes described herein. Examples of HMGR encoding or polypeptide sequences include SEQ ID Nos 7 to 10, or variants or fragments of these. Variants may be homologues, alleles, or artificial derivatives etc. as discussed in relation to QA-3-O-TriS genes or polypeptides as described above. For example an HMGR native to the host being utilised may be preferred - for example a yeast HMGR in a yeast host, and so on. HMGR genes are known in the art and may be selected, as appropriate in the light of the present disclosure.

***

It has also been reported that squalene synthase (SQS) is a potential rate-limiting step [19],

Thus one embodiment of the invention comprises the use of a heterologous SQS along with the QA-3-O-TriS genes and optionally HMGR described herein.

Examples of SQS encoding or polypeptide sequences include SEQ ID Nos 21 to 22, or variants or fragments of these. Variants may be homologues, alleles, or artificial derivatives etc. as discussed in relation to QA-3-O-TriS genes or polypeptides as described above. For example an SQS native to the host being utilised may be preferred - for example a yeast SQS in a yeast host, and so on. SQS genes are known in the art and may be selected, as appropriate in the light of the present disclosure.

When using certain hosts (for example yeasts) it may be desirable to introduce additional genes to improve the flux of QA production. Examples may include one or more plant cytochrome P450 reductases (CPRs) to serve as the redox partner to the introduced P450s. Thus one embodiment of the invention comprises the use of a heterologous cytochrome P450 reductase such as AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2) along with the QA polypeptides and QA-3-O-TriS genes described herein. Examples of AtATR2 encoding or polypeptide sequences include SEQ ID Nos 23 to 24, or variants or fragments of these. Variants may be homologues, alleles, or artificial derivatives etc. as discussed in relation to QA-3-O-TriS genes or polypeptides as described above.

Thus in one embodiment the nucleic acid utilised in the invention further encodes one or more of the following polypeptides:

(i) an HMG-CoA reductase (HMGR);

(ii) a squalene synthase (SQS)

wherein the HMGR or SQS are optionally selected from the respective polypeptides in Table 7 or substantially homologous variants or fragments of any of said polypeptides, or are encoded by the respective polynucleotides in Table 7, or substantially homologous variants or fragments of any of said polynucleotides.

It will be understood by those skilled in the art, in the light of the present disclosure, that additional genes may be utilised in the practice of the invention, to provide additional activities and\or improve expression or activity. These include those expressing co-factor or helper proteins, or other factors.

For brevity, unless context demands otherwise, any of these nucleic acid sequences (the “QA-3-O-TriS genes of the invention”, plus other genes effecting QA-3-O-TriS synthesis, or secondary modifications to QA-3-O-TriS) may be referred to herein as “QA-3-O-TriS- biosynthesis modifying nucleic acid”. Likewise the encoded polypeptides may be referred to herein as “QA-3-O-TriS-biosynthesis modifying polypeptides”.

It will be appreciated that where these generic terms are used in relation to any aspect or embodiment, the meaning or disclosure will be taken to apply mutatis mutandis to any of these sequences individually.

Vectors

As one aspect of the invention, there is disclosed a method employing the co-infiltration of a plurality of Agrobacterium tumefaciens strains each carrying one or more of the QA-3-O-TriS nucleic acids discussed above for concerted expression thereof in a biosynthetic pathway discussed above.

In some embodiments, at least 2 or 3 different Agrobacterium tumefaciens strains are co-infiltrated e.g. each carrying a QA-3-O-TriS nucleic acid.

The genes may be present from transient expression vectors.

A preferred expression system utilises the called “'Hyper-Translatable' Cowpea Mosaic Virus ('CPMV-HT') system, described in W02009/087391 the disclosure of which is specifically incorporated herein in support of the embodiments using the CPMV-HT system - for example vectors based on pEAQ-HT expression plasmids.

Thus the vectors (typically binary vectors) for use in the present invention will typically comprise an expression cassette comprising:

(i) a promoter, operably linked to

(ii) an enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated;

(iii) a QA-3-O-TriS nucleic acid sequence as described above;

(iv) a terminator sequence; and optionally

(v) a 3’ UTR located upstream of said terminator sequence.

Further examples of vectors and expression systems useful in the practice of the invention are described in more detail hereinafter.

Hosts

In aspects of the invention, a host may be converted from a phenotype whereby the host is unable to carry out effective QA-3-O-TriS biosynthesis from OS to a phenotype whereby the host is able to carry out said QA-3-O-TriS-biosynthesis, such that QA-3-O-TriS can be recovered therefrom or utilised in vivo to synthesize downstream products.

As explained above, QA biosynthesis can also be engineered into plants based on the disclosure of prior-filed unpublished PCT/EP2018/086430 (subsequently published as WO 2019/122259). Since the QA precursor (2,3-oxidosqualene) is ubiquitous in higher plants due to its role in sterol biosynthesis, the present invention has wide applicability in plant hosts. As discussed herein, additional activities may be employed when practising the invention in microorganisms.

Examples of hosts include plants such as Nicotiana benthamiana and microorganisms such as yeast. These are discussed in more detail below.

The invention may comprise transforming the host with heterologous nucleic acid as described above by introducing the QA-3-O-TriS nucleic acid into the host cell via a vector and causing or allowing recombination between the vector and the host cell genome to introduce a nucleic acid according to the present invention into the genome.

In another aspect of the invention, there is provided a host cell transformed with a heterologous nucleic acid which comprises a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have said QA-3-O-TriS-biosynthesis activity, wherein expression of said nucleic acid imparts on the transformed host the ability to carry out QA-3-O-TriS-biosynthesis from OS, or improves said ability in the host.

The invention further encompasses a host cell transformed with nucleic acid or a vector as described above (e.g. comprising the QA-3-O-TriS-biosynthesis modifying nucleotide sequences) especially a plant or a microbial cell. In the transgenic host cell (i.e. transgenic for the nucleic acid in question) the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. There may be more than one heterologous nucleotide sequence per haploid genome.

The methods and materials described herein can be used, inter alia, to generate stable crop-plants that accumulate QA-3-O-TriS. Examples of plants include row crops such as sunflower, potato, canola, dry bean, field pea, flax, safflower, buckwheat, cotton, maize, soybeans, and sugar beets. Major crop-plants such as corn, wheat, oilseed rape and rice may also be preferred hosts.

Plants which include a plant cell according to the invention are also provided.

Production of products

The methods described above may be used to generate glycosylated QA, such as QA-3-O-TriS, in a heterologous host. The glycosylated QA, such as QA-3-O-TriS, will generally be non-naturally occurring in the species into which they are introduced.

Glycosylated QA, such as QA-3-O-TriS, from the plants or methods of the invention may be isolated and commercially exploited.

The methods above may form a part of, possibly one step in, a method of producing downstream products, such as QS-21 in a host. The method may comprise the steps of culturing the host (where it is a microorganism) or growing the host (where it is a plant) and then harvesting it and purifying the glycosylated QA, such as QA-3-O-TriS, or a downstream product or derivative (e.g. QS-21) product therefrom. The product thus produced forms a further aspect of the present invention. The utility of QS-21 is described above.

Alternatively, glycosylated QA, such as QA-3-O-TriS, may be recovered to allow for further chemical synthesis of downstream compounds.

Novel genes of the invention

In support of the present invention, the present inventors have newly characterised or identified sequences from Q. saponaria which are believed to be involved in the synthesis of glycosylated compounds (see SEQ. ID: Nos 1-6; also 25-32).

In one embodiment (of this, or other aspects of the invention concerning these sequences), the sequences are selected from any of SEQ ID Nos 1-6.

In preferred embodiments, the methods of the present invention will include the use of one or more of these newly characterised QA-3-O-TriS nucleic acids of the invention (e.g. one, two, or three such QA-3-O-TriS nucleic acids) optionally in conjunction with the manipulation of other genes affecting QA biosynthesis known in the art.

These newly characterised QA-3-O-TriS sequences from Q. saponaria (SEQ. ID: Nos 1-6 also 25-32) form aspects of the invention in their own right, as do derived variants and materials of these sequences, and methods of using them.

Some aspects and embodiments of the present invention will now be described in more detail.

Detailed description of the invention

The present inventors utilised a variety of genome and transcriptome approaches with Q. saponaria to begin to elucidate biosynthetic pathways associated with glycosylation of QA. Functional characterisation of candidate genes by transient expression in Nicotiana benthamiana has led to the identification of three enzymes from Q. saponaria which together are capable of biosynthesis of QA-3-O-TriS from QA.

In different embodiments, the present invention provides means for manipulation of total levels of glycosylated QAs in host cells such as microorganisms or plants.

In one aspect of the present invention, the QA-3-O-TriS modifying nucleic acid described above is in the form of a recombinant and preferably replicable vector.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable, and which can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).

As is well known to those skilled in the art, a “binary vector” system includes (a) border sequences which permit the transfer of a desired nucleotide sequence into a plant cell genome; (b) desired nucleotide sequence itself, which will generally comprise an expression cassette of (i) a plant active promoter, operably linked to (ii) the target sequence and\or enhancer as appropriate. The desired nucleotide sequence is situated between the border sequences and is capable of being inserted into a plant genome under appropriate conditions. The binary vector system will generally require other sequence (derived from A. tumefaciens) to effect the integration. Generally this may be achieved by use of so called "agro-infiltration" which uses Agrobacterium-mediated transient transformation. Briefly, this technique is based on the property of Agrobacterium tumefaciens to transfer a portion of its DNA ("T-DNA") into a host cell where it may become integrated into nuclear DNA. The T-DNA is defined by left and right border sequences which are around 21-23 nucleotides in length. The infiltration may be achieved e.g. by syringe (in leaves) or vacuum (whole plants). In the present invention the border sequences will generally be included around the desired nucleotide sequence (the T-DNA) with the one or more vectors being introduced into the plant material by agro-infiltration.

Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression (e.g. for expressing a heterologous nucleic acid within a host or one or more cells of a host). Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sam brook etal, 1989, Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. higher plant, mosses, yeast or fungal cells).

A vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. yeast and bacterial, or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements (optionally in combination with a heterologous enhancer, such as the 35S enhancer discussed in the Examples below). The advantage of using a native promoter is that this may avoid pleiotropic responses. In the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell

By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA).

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.

In a preferred embodiment, the promoter is an inducible promoter.

The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no

expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.

Thus nucleic acid according to the invention may be placed under the control of an externally inducible gene promoter to place expression (expressing the heterologous sequence) under the control of the user. An advantage of introduction of a heterologous gene into a plant cell, particularly when the cell is comprised in a plant, is the ability to place expression of the gene under the control of a promoter of choice, in order to be able to influence gene expression, and therefore QA-3-O-TriS biosynthesis, according to preference. Furthermore, mutants and derivatives of the wild-type gene, e.g. with higher or lower activity than wild-type, may be used in place of the endogenous gene.

Thus this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter (optionally inducible) operably linked to a nucleotide sequence provided by the present invention, such as the QA-3-O-TriS-biosynthesis modifying gene, most preferably one of the QA-3-O-TriS nucleic acids which are described herein, or a derivative thereof.

Particularly of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Cray RRD ed.) Oxford, BIOS Scientific Publishers, pp 121-148). Suitable vectors may include plant viral-derived vectors (see e.g. EP-A- 194809).

Preferably the vectors of the present invention which are for use in plants comprise border sequences which permit the transfer and integration of the expression cassette into the plant genome. Preferably the construct is a plant binary vector. Preferably the binary transformation vector is based on pPZP (Hajdukiewicz, et al. 1994). Other example constructs include pBin19 (see Frisch, D. A., L. W. Harris-Haller, et al. (1995). “Complete Sequence of the binary vector Bin 19.” Plant Molecular Biology 27: 405-409).

Suitable promoters which operate in plants include the Cauliflower Mosaic Virus 35S (CaMV 35S). Other examples are disclosed at pg. 120 of Lindsey & Jones (1989) “Plant Biotechnology in Agriculture” Pub. OU Press, Milton Keynes, UK. The promoter may be selected to include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression. Inducible plant promoters include the ethanol induced promoter of Caddick et al (1998) Nature Biotechnology 16: 177-180.

If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to antibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate). Positive selection system such as that described by Haldrup et al. 1998 Plant molecular Biology 37, 287-296, may be used to make constructs that do not rely on antibiotics.

As explained above, a preferred vector is a 'CPMV-HT' vector as described in W02009/087391. The Examples below demonstrate the use of these pEAQ-HT expression plasmids.

These vectors (typically binary vectors) for use in the present invention will typically comprise an expression cassette comprising:

(i) a promoter, operably linked to

(ii) an enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated;

(iii) a QA-3-O-TriS nucleic acid sequence as described above;

(iv) a terminator sequence; and optionally

(v) a 3’ UTR located upstream of said terminator sequence.

“Enhancer” sequences (or enhancer elements), as referred to herein, are sequences derived from (or sharing homology with) the RNA-2 genome segment of a bipartite RNA virus, such as a comovirus, in which a target initiation site has been mutated. Such sequences can enhance downstream expression of a heterologous ORF to which they are attached.

Without limitation, it is believed that such sequences when present in transcribed RNA, can enhance translation of a heterologous ORF to which they are attached.

A “target initiation site” as referred to herein, is the initiation site (start codon) in a wild-type RNA-2 genome segment of a bipartite virus (e.g. a comovirus) from which the enhancer sequence in question is derived, which serves as the initiation site for the production (translation) of the longer of two carboxy coterminal proteins encoded by the wild-type RNA-2 genome segment.

Typically, the RNA virus will be a comovirus as described hereinbefore.

Most preferred vectors are the pEAQ vectors of W02009/087391 which permit direct cloning version by use of a polylinker between the 5’ leader and 3 UTRs of an expression cassette including a translational enhancer of the invention, positioned on a T-DNA which also contains a suppressor of gene silencing and an NPTII cassettes.

The presence of a suppressor of gene silencing in such gene expression systems is preferred but not essential. Suppressors of gene silencing are known in the art and described in WO/2007/135480. They include HcPro from Potato virus Y, H e-Pro from TEV, P19 from TBSV, rgsCam, B2 protein from FHV, the small coat protein of CPMV, and coat protein from TCV. A preferred suppressor when producing stable transgenic plants is the P19 suppressor incorporating a R43W mutation.

The present invention also provides methods comprising introduction of such a construct into a plant cell or a microbial (e.g. bacterial, yeast or fungal) cell and/or induction of expression of a construct within a plant cell, by application of a suitable stimulus e.g. an effective exogenous inducer.

As an alternative to microorganisms, cell suspension cultures of engineered glycosylated QA -producing plant species, including also the moss Physcomitrella patens, may be cultured in

fermentation tanks (see e.g. Grotewold et al. (Engineering Secondary Metabolites in Maize Cells by Ectopic Expression of Transcription Factors, Plant Cell, 10, 721-740, 1998).

In a further aspect of the invention, there is disclosed a host cell containing a heterologous construct according to the present invention, especially a plant or a microbial cell.

The discussion of host cells above in relation to reconstitution of QA-3-O-TriS biosynthesis in heterologous organisms applies mutatis mutandis here.

Thus a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a construct as described above into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce a nucleic acid according to the present invention into the genome.

The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention (e.g. comprising the QA-3-O-TriS -biosynthesis modifying nucleotide sequence) especially a plant or a microbial cell. In the transgenic plant cell (i.e. transgenic for the nucleic acid in question) the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. There may be more than one heterologous nucleotide sequence per haploid genome.

Yeast has seen extensive employment as a triterpene-producing host and is therefore potentially well adapted for QA and then QA-3-O-TriS biosynthesis.

Therefore, in one embodiment, the host is a yeast. For such hosts, it may be desirable to introduce additional genes to improve the flux of QA, and hence QA-3-O-TriS production as described above. Examples may include one or more plant cytochrome P450 reductases (CPRs) to serve as the redox partner to the introduced P450s, as well as an HMGR. It may likewise be desirable to introduce additional genes to contribute other elements of the QA or improve QA-3-O-TriS pathways. These may include enzymes providing UDP-sugar donors and the like (see e.g. Ohashi T, Hasegawa Y, Misaki R, Fujiyama K (2016) “Substrate preference of citrus naringenin rhamnosyltransferases and their application to flavonoid glycoside production in fission yeast”. (2016). Applied Microbiology and Biotechnology.

100(2): 687-696.); Oka T, Jigami Y. (2006). “Reconstruction of de novo pathway for synthesis of UDP-glucuronic acid and U DP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae”. FEBS J.273(12):2645-57). In the light of the present disclosure, those skilled in the art can provide such ancillary activities as required.

Plants, which include a plant cell transformed as described above, form a further aspect of the invention.

If desired, following transformation of a plant cell, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

In addition to the regenerated plant, the present invention embraces all of the following: a clone of such a plant, seed, selfed or hybrid progeny and descendants (e.g. F1 and F2 descendants). The invention also provides a plant propagule from such plants, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. In all cases these plants or parts include the plant cell or heterologous QA-3-O-TriS-biosynthesis modifying DNA described above, for example as introduced into an ancestor plant.

It also provides any part of these plants (e.g. leaf, stem, dried or ground product, edible portion etc.), which in all cases include the plant cell or heterologous QA-3-O-TriS-biosynthesis modifying DNA described above.

The present invention also encompasses the expression product of any of the coding QA-3-O-TriS -biosynthesis modifying nucleic acid sequences disclosed and methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions, which may be in suitable host cells.

As described below, plant backgrounds such as those above may be natural or transgenic e.g. for one or more other genes relating to glycosylated QA, such as QA-3-O-TriS, biosynthesis, or otherwise affecting that phenotype or trait.

In modifying the host phenotypes, the QA-3-O-TriS nucleic acids described herein may be used in combination with any other gene, such as transgenes affecting the rate or yield of QA-3-O-TriS, or its modification, or any other phenotypic trait or desirable property.

By use of a combination of genes, plants or microorganisms (e.g. bacteria, yeasts or fungi) can be tailored to enhance production of desirable precursors, or reduce undesirable metabolism.

As an alternative, down-regulation of genes in the host may be desired e.g. to reduce undesirable metabolism or fluxes which might impact on QA-3-O-TriS yield.

Such down regulation may be achieved by methods known in the art, for example using anti-sense technology.

In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.

An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, and US-A-5,231,020. Further refinements of the gene silencing or co-suppression technology may be found in W095/34668 (Biosource); Angell & Baulcombe (1997) The EMBO Journal 16,12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553.

Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire A. et al Nature, Vol 391, (1998)). dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) (See also Fire (1999) Trends Genet. 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490, Hammond et al. (2001) Nature Rev. Genes 2: 1110-1119 and Tuschl (2001) Chem. Biochem. 2: 239-245).

RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt) The siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001)

Another methodology known in the art for down-regulation of target sequences is the use of “microRNA” (miRNA) e.g. as described by Schwab et al 2006, Plant Cell 18, 1121-1133.

This technology employs artificial miRNAs, which may be encoded by stem loop precursors incorporating suitable oligonucleotide sequences, which sequences can be generated using well defined rules in the light of the disclosure herein.

Thus in one aspect, the invention provides a method for influencing or affecting glycosylated QA biosynthesis in a host, which method comprises any of the following steps of:

(i) causing or allowing transcription from a nucleic acid comprising the complement sequence of a QA-3-O-TriS nucleotide sequence such as to reduce the respective encoded polypeptide activity by an antisense mechanism;

(ii) causing or allowing transcription from a nucleic acid encoding a stem loop precursor comprising 20-25 nucleotides, optionally including one or more mismatches, of a QA-3-O-TriS nucleotide sequence such as to reduce the respective encoded polypeptide activity by an miRNA mechanism;

(iii) causing or allowing transcription from nucleic acid encoding double stranded RNA corresponding to 20-25 nucleotides, optionally including one or more mismatches, of a QA-3-O-TriS nucleotide sequence such as to reduce the respective encoded polypeptide activity by an siRNA mechanism.

The methods of the present invention embrace both the in vitro and in vivo production, or manipulation, of one or more glycosylated QAs. For example, QA-3-O-TriS polypeptides may be employed in fermentation via expression in microorganisms such as e.g. E.coli, yeast and filamentous fungi and so on. In one embodiment, one or more newly characterised QA-3-O-TriS sequences of the present invention may be used in these organisms in conjunction with one or more other biosynthetic genes.

In vivo methods are described extensively above, and generally involve the step of causing or allowing the transcription of, and then translation from, a recombinant nucleic acid molecule encoding the QA-3-O-TriS polypeptides.

In other aspects of the invention, the QA-3-O-TriS polypeptides (enzymes) may be used in vitro, for example in isolated, purified, or semi-purified form. Optionally they may be the product of expression of a recombinant nucleic acid molecule.

Newly characterised sequences from Q. saponaria

As noted above, in support of the present invention, the inventors have identified genes from Q. saponaria which are believed to encode polypeptides which affect QA-3-O-TriS biosynthesis (see SEQ. ID: Nos 1-6 and 25-32 in Table 5).

The above newly characterised QA-3-O-TriS biosynthetic genes from Q. saponaria thus form aspects of the present invention in their own right.

In a further aspect of the present invention, there are disclosed nucleic acids which are variants of the QA-3-O-TriS nucleic acid derived from Q. saponaria discussed above.

Such variants, as with the native genes discussed herein, may be used to alter the glycosylated QA (e.g. QA-3-O-TriS) content of a plant, as assessed by the methods disclosed herein. For instance, a variant nucleic acid may include a sequence encoding a variant QA-3-O-TriS polypeptide sharing the relevant biological activity of the native QA-3-O-TriS polypeptide, as discussed above. Examples include variants of any of SEQ ID Nos 2,

4, 6, 26, 28, 30, or 32.

Derivatives

Described herein are methods of producing a derivative nucleic acid comprising the step of modifying any of the QA-3-O-TriS genes of the present invention disclosed above, particularly the QA-3-O-TriS sequences from Q. saponaria.

Changes may be desirable for a number of reasons. For instance, they may introduce or remove restriction endonuclease sites or alter codon usage. This may be particularly desirable where the QA-3-O-TriS genes are to be expressed in alternative hosts e.g. microbial hosts such as yeast. Methods of codon optimizing genes for this purpose are known in the art (see e.g. Elena, Claudia, et al. "Expression of codon optimized genes in microbial systems: current industrial applications and perspectives." Frontiers in microbiology 5 (2014)). Thus sequences described herein including codon modifications to maximise yeast expression represent specific embodiments of the invention.

Alternatively, changes to a sequence may produce a derivative by way of one or more (e.g. several) of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more (e.g. several) amino acids in the encoded polypeptide.

Such changes may modify sites which are required for post translation modification such as cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for phosphorylation etc. Leader or other targeting sequences (e.g. membrane or golgi locating sequences) may be added to the expressed protein to determine its location following expression if it is desired to isolate it from a microbial system.

Other desirable mutations may be random or site-directed mutagenesis in order to alter the activity (e.g. specificity) or stability of the encoded polypeptide. Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation. Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.

Fragments

The present invention may utilise fragments of the genes encoding the QA-3-O-TriS polypeptides of the present invention disclosed above, particularly the QA-3-O-TriS sequences from Q. saponaria.

Thus the present invention provides for the production and use of fragments of the full-length QA-3-O-TriS polypeptides of the invention disclosed herein, especially active portions thereof. An “active portion” of a polypeptide means a peptide which is less than said full length polypeptide, but which retains its essential biological activity e.g. in relation to glycosylation of QA (see e.g. Table 5).

A “fragment” of a polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids. Fragments of the polypeptides may include one or more epitopes useful for raising antibodies to a portion of any of the amino acid sequences disclosed herein. Preferred epitopes are those to which antibodies are able to bind specifically, which may be taken to be binding a polypeptide or fragment thereof of the invention with an affinity which is at least about 1000x that of other polypeptides.

For brevity, and of these QA-3-O-TriS sequences from the Q. saponaria, or variants (e.g. derivatives such as fragments thereof) may be referred to as “Qs QA-3-O-TriS sequences (or nucleic acid, or polypeptide)”. These Qs QA-3-O-TriS polypeptides, and nucleic acids encoding them, form one aspect of the invention.

It will be appreciated that, where this term is used generally, it also applies to any of these sequences individually.

Thus in one aspect of the invention, there is disclosed isolated nucleic acid encoding any of these polypeptides (2, 4 6, , 26, 28, 30, or 32). Preferably, this may have the sequence of 1, 3, 5, 25, 27, 29, or 31. Other nucleic acids of the invention include those which are degeneratively equivalent to these, or homologous variants (e.g. derivatives) of these.

Aspects of the invention further embrace isolated nucleic acid comprising a sequence which is complementary to any of those discussed hereinafter.

In vitro or in vivo use of a QA-3-O-TriS sequence to catalyse its respective biological activity (QA-glycosylation, for example as described in Fig. 3 or Figure 9 or Table 5) forms another aspect of the invention.

Thus the invention further provides a method of influencing or affecting glycosylated QA (e.g. QA-3-O-TriS) biosynthesis in a host such as a plant, the method including causing or allowing transcription of a heterologous Qs QA-3-O-TriS nucleic acid as discussed above within the cells of the plant. The step may be preceded by the earlier step of introduction of the QsQA-3-O-TriS nucleic acid into a cell of the plant or an ancestor thereof.

Such methods will usually form a part of, possibly one step in, a method of producing a glycosylated QA (e.g. QA-3-O-TriS) in a host such as a plant. Preferably the method will employ a Qs QA-3-O-TriS polypeptide of the present invention (e.g. in Table 5) or derivative thereof, as described above, or nucleic acid encoding either.

In a further embodiment, there are provided antibodies raised to a Qs QA-3-O-TriS polypeptide or peptide of the invention

Some aspects of the invention as it relates to heterologous reconstitution of the biosynthetic pathways discussed above will now be discussed in more detail.

“Nucleic acid” according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid). Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted forT where it occurs, is encompassed. Nucleic acids may include more than one nucleic acid molecule. Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin, and double or single stranded. Where used herein, the term “isolated” encompasses all of these possibilities. The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Nucleic acids may comprise, consist, or consist essentially of, any of the sequences discussed hereinafter.

The “complement” of a nucleic acid described herein means the complementary sequence of the or a nucleotide sequence comprised by the nucleic acid. Optionally complementary sequences are full length compared to the reference nucleotide sequence.

The term "heterologous" is used broadly herein to indicate that the gene/sequence of nucleotides in question (e.g. encoding QA-3-O-TriS -biosynthesis modifying polypeptides) have been introduced into said cells of the host or an ancestor thereof, using genetic engineering, i.e. by human intervention. Nucleic acid heterologous to a host cell will be non-naturally occurring in cells of that type, variety or species. Thus the heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant. A further possibility is for a nucleic acid sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.

“Transformed” in this context means that the nucleotide sequences of the heterologous nucleic acid alter one or more of the cell’s characteristics and hence phenotype e.g. with respect to glycosylated QA e.g. QA-3-O-TriS biosynthesis. Such transformation may be transient or stable.

“Unable to carry out QA-3-O-TriS biosynthesis” means that the host, prior to the conversion, does not, or is not believed to, naturally produce detectable or recoverable levels of QA-3-O-TriS under normal metabolic circumstances of that host. Following the application of the invention it is able to produce detectable or recoverable levels of QA-3-O-TriS.

The nucleotide sequence information provided herein may be used to design probes and primers for probing or amplification. An oligonucleotide for use in probing or PCR may be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use in processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length. Small variations may be introduced into the sequence to produce ‘consensus’ or ‘degenerate’ primers if required.

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the single stranded DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells. Probing may optionally be done by means of so-called ‘nucleic acid chips’ (see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review).

In one embodiment, a variant encoding a QA-3-O-TriS -biosynthesis modifying polypeptide in accordance with the present invention is obtainable by means of a method which includes:

(a) providing a preparation of nucleic acid, e.g. from plant cells. Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc.), such as are described hereinafter,

(b) providing a nucleic acid molecule which is a probe or primer as discussed above,

(c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and,

(d) identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule. Binding of a probe to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include amplification using PCR (see below), RN’ase cleavage and allele specific oligonucleotide probing. The identification of successful hybridisation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification of a vector in a suitable host.

Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further.

For example, hybridizations may be performed, according to the method of Sambrook et al. (below) using a hybridization solution comprising: 5X SSC (wherein ‘SSC’ = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7), 5X Denhardt’s reagent, 0.5- 1.0% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1 % SDS; (3) 30 minutes - 1 hour at 37°C in 1X SSC and 1% SDS; (4) 2 hours at 42-65°C in 1X SSC and 1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989): Tm = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [Na+] = [0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57°C. The Tm of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.

It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Other suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% d extra n sulfate and a final wash at 55°C in 0.1X SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% d extra n sulfate and a final wash at 60°C in 0.1X SSC, 0.1 % SDS.

In a further embodiment, hybridization of a nucleic acid molecule to a variant may be determined or identified indirectly, e.g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR). PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of a QA-3-O-TriS gene of the present invention are employed. Using RACE PCR, only one such primer may be needed (see "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990)).

Thus a method involving use of PCR in obtaining nucleic acid according to the present invention may include:

(a) providing a preparation of plant nucleic acid, e.g. from a seed or other appropriate tissue or organ,

(b) providing a pair of nucleic acid molecule primers useful in (i.e. suitable for) PCR, at least one of said primers being a primer according to the present invention as discussed above,

(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR,

(d) performing PCR and determining the presence or absence of an amplified PCR product. The presence of an amplified PCR product may indicate identification of a variant.

In all cases above, if need be, clones or fragments identified in the search can be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.

Purified protein (polypeptide, enzyme), or a fragment, mutant, derivative or variant thereof, e.g. produced recombinantly by expression from encoding nucleic acid therefor, forms one aspect of the invention.

Such purified polypeptides may be used to raise antibodies employing techniques which are standard in the art. Antibodies and polypeptides comprising antigen-binding fragments of antibodies may be used in identifying homologues from other species as discussed further below.

Methods of producing antibodies include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82). Antibodies may be polyclonal or monoclonal.

As an alternative or supplement to immunising a mammal, antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous

bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.

Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then the encoding genes.

Antibodies may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic.

***

A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

Abbreviations

Compound 1 - 3β-{[α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β- D -glucopyranosiduronic acid]oxy}-quillaic acid

Compound 2 - 3β-{[β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid]oxy}-quillaic acid

Galp- D-Galactopyranose

GlcpA - D-Glucopyranuronic acid (Additional numbers denote specific carbons i.e. GlcA-1) GmSGT2/GmUGT73P2 - Glycine max (soybean) Soyasaponin β-D-galactosyltransferase GTs - Glycosyltransferases

QA - Quillaic acid

QA-GlcpA - 3β-{[β-D-glucopyranosiduronic acid]oxy}-quillaic acid

QA-GlcpA-Galp - 3β-{[β-D-galactopyranosyl-(1->2)-β-D-glucopyranosiduronic acid]oxy}-quillaic acid

QA-GlcpA-[Galp]-Rhap - 3β-{[α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β- D -glucopyranosiduronic acid]oxy}-quillaic acid

QA-GlcpA-[Galp]-Xylp- 3β-{[β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid]oxy}-quillaic acid

QA-3-O-TriS - QA glycosylated at 3-O position with a branched trisaccharide which is either

QA-GlcpA-[Galp]-Rhap or QA-GlcpA-[Galp]-Xylp

OS - 2,3-oxidosqualene

***

QA-GlcAT - QA 3-O glucuronosyl transferase

QA-GalT - QA-GlcpA galactosyl transferase

QA-RhaT/XylT - QA-GlcpA-Galp Rhamnosyl and/or Xylosyl transferase

QsbAS - Q. saponaria β-amyrin synthase

Qs-3-O-GalT - Q. saponaria QA-GlcpA β-1,2-D-galactosyltransferase

QsCSL1 - Q. saponaria cellulose synthase-like enzyme (quillaic acid 3-O-glucuronosyltransferase)

QsCslG2 - Q. saponaria cellulose synthase-like enzyme (quillaic acid 3-O-glucuronosyltransferase)

Qs-3-O-RhaT/XylT - Q. saponaria QA-GlcpA-Galp dual β-1,3-D-xylosyltransferase/α-1,3-L-rhamnosyltransferase

Qs_0283850 - Q. saponaria QA-GlcpA-Galp α-1,3-L-rhamnosyltransferase DN20529_c0_g2_i8 - Q. saponaria QA-GlcpA-Galp α-1,3-L-rhamnosyltransferase

Qs 0283870 - Q. saponaria QA-GlcpA-Galp β-1,3-D-xylosyltransferase

QsCYP716-C-28 - Q. saponaria quillaic acid C-28 oxidase

QsCYP716-C-16α - Q. saponaria quillaic acid C-16α oxidase

QsCYP714-C-23 - Q. saponaria quillaic acid C-23 oxidase

Rhap - L-Rhamnopyranose

tHMGR - A vena strigosa (diploid oat) truncated 3-hydroxy, 3-methylbutyryl-CoA reductase Family 1 UGT - Family 1 UDP-dependent glycosyltransferase

Xylp - D-Xylopyranose

Qs_2Q73886_D6 - synonymous with Qs-3-O-GalT

Qs_2015879_D7 - synomymous with Qs-3-O-RhaT/XylT

Figures

Figure 1: QS-21.The major structural features are highlighted, including the quillaic acid triterpene aglycone, a branched trisaccharide at C-3, linear tetrasaccharide at C-28 and an arabinosylated 18-carbon acyl chain attached to the β-D-fucose at C-28.

Figure 2: Production of quillaic acid from 2,3-oxidosqualene via β-amyrin. Numbering of important β-amyrin carbons referred to herein are labelled in red. The pathway from β-amyrin requires oxidation at three (C-28, C-23 and C-16α) positions. These oxidation steps are shown in a linear fashion for simplicity, however they could occur in any order.

Figure 3: Production of quillaic acid trisaccharide derivative (QA-3-O-TriS) from quillaic acid. The pathway (showed in linear sequential form for simplicity) entails a 3-step glycosylation at C-3 of quillaic acid beginning with D-Glucuronic acid (GlcpA). GlcpA is further glycosylated with a β-1,2-D-Galactose (Galp) and with a β-1,3-O-Xylose (Xylp), in one or other order.

Figure 4: Mining for candidate QS-21 UDP-dependent glycosyltransferases (UGTs).

Phylogenetic tree of Quillaja saponaria UGT candidates (red) with characterised UGTs from other plant species (black) (listed in Table 3). Functionally characterised triterpene UGTs are indicated in blue. Q. saponaria UGTs whose genes are predicted to be within biosynthetic gene clusters (BCGs) are indicated by asterisks. The UGT phylogenetic groups (Groups A-P) are labelled as described in Ross, J., Li, Y., Lim, E., and Bowles, D. J. (2001). “Higher plant glycosyltransferases”. Genome Biol., 2:REVIEWS3004, and Caputi, L, Malnoy, M., Goremykin, V., Nikiforova, S., and Martens, S. (2012). “A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land”. Plant J., 69:1030-42], The tree was constructed using the Neighbour Joining method with 1000 bootstrap replicates (% support for branch points is shown). The scale bar shows 0.1 substitutions per site at the amino acid level. The Q. saponaria contigs in the 1 KP database consist of a 4-letter code (OQHZ) followed by seven digits; this seven digit code is included in the name for the candidate UGT genes.

Figure 5: Conversion of quillaic acid by QsCSLI. Accumulation of quillaic acid was detected in leaves expressing QsbAS and C-28/C-23/C-16α oxidases. The addition of Q. saponaria Cellulose Synthase-like (QsCSLI) resulted in lower levels of quillaic acid and the accumulation of a new peak with the mass of quillaic acid with the addition of a glucuronide residue [m/z = 661, retention time = 13.9 min]. IS = internal standard (digitoxin).

Figure 6: GmUGT73P2 (accession number: BAI 99584) from Glycine max catalyses the addition of D-galactose to soyasapogenol B monoglucuronide with a β-1,2-linkage to form soyasaponin III (Shibuya et al., 2010).

Figure 7: Conversion of QA-GlcpA by Qs_2073886_D6 (Qs-3-O-GalT). Co-expression of Qs_2073886_D6 with genes required for production of the putative QA-GlcpA

( tHMGR/QsbAS/C YP716-C-28/C YP716-C-16a/C YP714-C23/QsCSL1) resulted in the appearance of a more polar peak with the expected mass of QA-GlcpA with the addition of a hexose [m/z = 823, retention time = 12.6 min]. The average mass spectrum of this peak is shown. Co-expression of genes required for production of the putative QA-GlcpA with GmUGT73P2 resulted in a new peak with the same retention time as the Qs_2073886_D6 product. IS = internal standard (digitoxin).

Figure 8: Co-expression of Qs_2015879_D7 with genes required for production of the putative QA-GlcpA-Galp ( tHMGR/QsbA S/C YP716-C-28/CYP716-C-16a/CYP714-C23/QsCSL1/Qs-3-O-GalT) resulted in the appearance of two peaks that co-eluted with

similar retention times as the putative QA-GlcpA-Galp peak. The more polar peak had the expected mass of QA-GlcpA-Galp with the addition of a deoxyhexose [m/z = 869, retention time = 12.5 min] and the less polar peak had the expected mass of QA-GlcpA-Galp with the addition of a pentose [m/z = 855, retention time = 12.75 min]. The average mass spectrum of each peak and predicted structures are shown. Co-expression of a combination without the Qs-3-O-GalT gene did not result in any new peaks suggesting that Qs_2015879_D7 is dependent on Qs-3-O-GalT activity. IS = internal standard (digitoxin).

Figure 9: Proposed biosynthesis of QA-GlcpA-[Galp]-Rhap and QA-GlcpA-[Galp]-Xylp from quillaic acid with the enzymes characterised herein.

Figure 10: Structure of isolated compounds 1 and 2.

Figure 11: Full 13C NMR assignment and Key HMBC (H→C) reported for compounds 1 and 2

Figure 12: Schematic for isolation of the trisaccharides from N. benthamiana leaf material.

Figure 13: Semi-preparative HPLC chromatogram for purification of compounds 1 and 2.

Figure 14: Heatmap showing the expression profiles of the previously characterised QS-21 biosynthetic genes.

Figure 15: Phylogenetic tree of Quillaja saponaria cellulose synthase superfamily proteins (bold) with cellulose synthase superfamily proteins from Oryza sativa and Arabidopsis thaliana. The tree was constructed using the Neighbour Joining method with 1000 bootstrap replicates (% support for branch points is shown). The Q. saponaria genes in the genomic database consist of a code (QUISA32244_EIv1_) followed by seven digits; this seven-digit code is included in the name for the Q. saponaria proteins; Q. saponaria proteins in the same subfamily (CsIG) as CSL1 have been renamed CslG2-6.

Figure 16: Heatmap showing the expression profiles of the previously characterised QS-21 biosynthetic genes and CslG2-CslG6.

Figure 17: Co-expression of CslG2 with genes r used for production of quillaic acid (tHMGR/QsbAS/CYP716-C-28/CYP716-C-16a/CYP714-C23) resulted in the reduction of the quillaic acid peak [m/z = 485, retention time = 19.6 min] and the appearance of a more polar peak with the expected mass of quillaic acid with the addition of a glucuronide residue and the same retention time as the CSL1 product [m/z = 661, retention time = 14.0 min]. IS = internal standard (digitoxin).

Figure 18: Qs-3-O-RhaT/XylT is a chimera between two adjacent genes, Qs_0283860 and Qs_0283870 in the Quillaja saponaria genome. The Q. saponaria genes in the genome database consist of a code (QUISA32244_EIv1) followed by seven digits; this seven-digit code is included in the name for the Q. saponaria genes.

Figure 19: Conversion of QA-GlcpA-Galp by Qs_0283850, DN20529_c0_g2_i8 and Qs_0283870. Co-expression of the dual function Qs-3-O-RhaT/XylT with the genes used for production of QA-GlcpA-Galp (tHMGR/QsbAS/CYP716-C-28/CYP716-C-16a/CYP714-C23/QsCSL1/Qs-3-O-GalT) results in the conversion of QA-GlcpA-Galp (retention time = 12.6 min, MW = 824) to QA-GlcpA-[Galp]-Rhap (retention time = 12.5 min, MW = 970) and QA-GlcpA-[Galp]-Xylp (retention time = 12.75 min, MW = 956). Co-expression of Qs_0283850 or DN20529_c0_g2_i8 with the genes required for production of QA-GlcpA-Galp resulted in the reduction of the QA-GlcpA-Galp peak at 12.6 minutes and the appearance of a single new peak, with the same retention time (12.5 min) and molecular weight (MW = 970) as QA-GlcpA-[Galp]-Rhap. Co-expression of Qs_0283870 with the genes required for production of QA-GlcpA-Galp resulted in the reduction of the QA-GlcpA-Galp peak at 12.6 min and the accumulation of a new single peak with the same retention time (12.75 min) and molecular weight (MW = 956) as QA-GlcpA-[Galp]-Xylp.