transferase

Abstract

The present invention relates generally to genes and polypeptides useful for glycosylating quillaic acids in a host cell, including enzymes capable of sequential glycosylation at the C-3 position of quillaic acids. The present invention further relates to systems, methods, and products of using the genes and polypeptides.

Description

Technical Field

[0001] This invention generally relates to genes and peptides that can be used to modify saponins in host cells. The invention further relates to systems, methods, and products using said genes and peptides. Background Technology

[0002] Plants produce a variety of cyclic triterpenes, such as sterols and triterpenoids, which are the main products of the mevalonate pathway.

[0003] QS-21 is a complex triterpenoid saponin synthesized from the Chilean saponaria (Quillaja saponaria) (Fabales) (Figure 1). QS-21 is a potent immunostimulant that enhances both antibody and specific T-cell responses, making it a significant adjuvant potential [1,2]. AS01 adjuvant is a liposomal formulation of QS-21 and 3-O-deacylated-4'-monophosphoryl lipid A and is currently licensed as part of GlaxoSmithKline's "Shingrix" vaccine against shingles and "Mosquirix" vaccine against malaria [1,3]. As these represent the first licensed human vaccines using QS-21, demand for this molecule is expected to increase significantly in the coming years.

[0004] Currently, QS-21 is of natural origin. Engineering the production of QS-21 (or a suitable semi-synthetic intermediate) in a heterologous host would provide an alternative or complement to current production methods. However, very little is known about the biosynthesis of QS-21, which presents a significant obstacle to achieving this goal.

[0005] Biochemically, QS-21 consists of a C-30 triterpenoid skeleton called saponin. This skeleton is decorated with a branched trisaccharide at C-3 and a linear tetrasaccharide at C-28. The terminal sugar of the tetrasaccharide can be β-D-apiose or β-D-xylose. Finally, the β-D-fucose in the tetrasaccharide is characterized by a C-18 acyl chain glycosylated with arabinose (Figure 1).

[0006] The biosynthesis of saponin acid begins with β-amyrin, which is synthesized by cyclizing the universal linear precursor 2,3-oxidized squalene (OS) using an oxidized squalene cyclase (OSC) (Figure 2). The β-amyrin scaffold is further oxidized at C-28, C-16α, and C-23 by carboxylic acids, alcohols, and aldehydes, respectively, to form saponin acid (QA). Figure 2 illustrates the proposed biosynthetic pathway for this purpose.

[0007] The previously submitted, unpublished PCT / EP2018 / 086430 (later published as WO 2019 / 122259) describes the identification of enzymes involved in QA production. Candidate enzymes were cloned from leaf cDNA. By testing the enzymes through transient co-expression in *Nicotiana benthamiana*, one OSC and three cytochrome P450s (P450s) required for the synthesis of β-amyrin and its oxidation to saponin were identified (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.

[0008] Figure 3 shows a proposed scheme for glycosylation of saponin acid to 3β-{[β-D-xylanosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponin acid (QA-GlcpA-[Galp]-Xylp).

[0009] Some enzymes that have been reported to be able to glycosylate triterpenoids have previously been obtained from plants other than soapberry (Q. saponaria), including C-3 glucuronosyltransferase from licorice [Xu, G. et al., A novel glucuronosyltransferase has an unprecedented ability to catalyze continuous two-step glucuronosylation of glycyrrhetinic acid to yield glycyrrhizin. New Phytologist, 2016. 212(1): 123-135] and 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): 2258-64].

[0010] US 20190059314 and WO / 2020 / 049572 relate to genes (including regulatory and enzyme-encoding genes) reportedly available for use in the biosynthesis of steroidal alkaloids and saponins, and their use in altering the content of steroidal (sugar) alkaloids or phytosterols in plants. US 20190059314 discusses the use of genes encoding cellulose synthase-like proteins in this context.

[0011] However, no glycosyltransferases (GTs) responsible for glycosylation or sequential glycosylation at the C-3 position of QA have been previously reported. In summary, it is clear that providing such GTs would contribute to the field. Summary of the Invention

[0012] This invention relates to the identification of glycosyltransferases (GTs) responsible for continuous glycosylation at the C-3 position of QA.

[0013] As shown in Figure 3, the branched trisaccharide chain at the C-3 position in QS-21 is initiated by a β-D-glucuronic acid (GlcpA) residue attached to the 3-O position of QA. Then, the GlcpA residue is linked to D-galactose (Galp) via a β-1->2 bond and to D-xylose (Xylp) via a β-1,3 bond.

[0014] Please note that, for simplicity, the last two steps (the connection of Galp and Xylp to GlcpA) are shown in a linear fashion in Figure 3, but they may occur in any order.

[0015] Furthermore, as explained below, β-D-Xylp residues can be replaced by α-L-rhamnose residues in certain circumstances.

[0016] Specifically, the inventors have characterized two glucuronyltransferases, one galactosyltransferase, and rhamnosyltransferase, xylosyltransferase, and a dual rhamnosyl / xylosyltransferase derived from soap trees, which allow the 3-O position of QA to be glycosylated by the respective sugars within the 3-O-branched trisaccharide. Therefore, in the context of this invention, the number "3" in the term "3-O-branched trisaccharide" should be understood as the position C-3 of QA (as shown in Figure 2).

[0017] They named these enzymes “QsCSL1” and “QsCSLG2” (glucuronyltransferase), “Qs-3-O-GalT” (galactosyltransferase), “Qs_0283850”, “DN20529_c0_g2_i8”, “Qs_0283870” and “Qs-3-O-RhaT / XylT” (rhamnosyl and / or xylosyltransferase), as provided in Table 5.

[0018] Surprisingly, glucuronyltransferase is a “cellulose synthase-like” enzyme. Cellulose synthases are generally involved in cell wall biosynthesis, and the functions of most of its members are unknown

[13] .

[0019] Therefore, this disclosure provides new uses for such enzymes in QA glycosylation (in vivo or in vitro). The term "QA glycosylation" as used herein is widely used to further include the sequential glycosylation of already glycosylated QA.

[0020] ***

[0021] Galactosyltransferases and rhamnosyl / xylsyltransferases are a family of UDP-dependent glycosyltransferases (UGTs). These enzymes have previously been identified as involved in plant-specific glycosylation, although other enzymes have been found to play roles in the biosynthesis of plant-specific metabolites [8-12].

[0022] Figure 9 illustrates the synergistic operation of the enzymes (again depicted linearly for simplicity).

[0023] Therefore, the present invention provides a host cell or organism (e.g., a plant or microorganism) engineered to have the ability to biosynthesize glycosylated QA (e.g., a 3-O branched trisaccharide saponin (QA) derivative (“QA-3-O-TriS”)). Alternatively, the enzyme can be used in vitro to perform the corresponding activities reported in Table 5.

[0024] They can be advantageously used in combination with enzymes that allow the synthesis of QA from OS (e.g., characterized in the previously submitted, unpublished PCT / EP2018 / 086430 (later published as WO 2019 / 122259), the entire disclosure of which is expressly incorporated herein by cross-reference. Examples of such enzymes (defined as “QA polypeptides”) are shown in Table 8.

[0025] ***

[0026] Therefore, in one aspect of the invention, a host phenotype is provided that transforms the host from one incapable of biosynthesizing 3-O branched-chain trisaccharide saponin acid (“QA”) derivatives (“QA-3-O-TriS”).

[0027] QA-3-O-TriS is 3β-{[β-D-xylanopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponin acid (QA-GlcpA-[Galp]-Xylp) or (3β-{[α-L-rhamnosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponin acid)(QA-GlcpA-[Galp]-Rhap).

[0028] A method for converting a host phenotype capable of performing the QA-3-O-TriS biosynthesis by glycosylation at the 3-O position of QA.

[0029] The method includes, following an earlier step of introducing a heterologous nucleic acid into the host or its ancestor, a step of expressing the heterologous nucleic acid within the host or one or more of its cells.

[0030] The heteronucleotide comprises multiple nucleotide sequences, each of which encodes a polypeptide having the QA-3-O-TriS biosynthetic activity in combination.

[0031] For example, using the illustrative scheme of Figure 3, the encoded polypeptide (enzyme) may comprise any one or more of the following:

[0032] (i) QA 3-O glucuronyl transferase (“QA-GlcAT”), which is able to transfer D-glucuronic acid (“GlcpA”) to the 3-O position of saponin to form 3β-{[β-D-glucanyl pyranoside]oxy}-saponin (“QA-GlcpA”);

[0033] (ii) QA-GlcpA galactosyltransferase (“QA-GalT”), which is able to transfer D-galactose (“Galp”) to QA-GlcpA via a β-1->2 bond to form 3β-{[β-D-galactopyranosyl-(1->2)-β-D-glucopyranoside]oxy}-saponin (“QA-GlcpA-Galp”);

[0034] (iii) QA-GlcpA-Galp rhamnosyl / xylsyltransferase (“QA-RhaT / XylT”),

[0035] It can transfer L-rhamnose (“Rhap”) and / or D-xylose (“Xylp”) via 1->3 linkage to QA-GlcpA-Galp to form, respectively

[0036] (3β-{[α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponic acid)(“QA-GlcpA-[Galp]-Rhap”) and / or

[0037] 3β-{[β-D-xylanopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponin acid (“QA-GlcpA-[Galp]-Xylp”).

[0038] In one implementation, the QA-GlcpA-Galp rhamnosyl / xylosyltransferase can be a dual QA-GlcpA-Galp rhamnosyl / xylosyltransferase.

[0039] Each of the polypeptide or nucleotide sequences may optionally be obtained from or derived from the soap tree.

[0040] As non-limiting examples, QA-GlcAT, QA-GalT, and QA-RhaT / XylT are selected from the corresponding enzymes in Table 5 or Table 6, or substantially homologous variants or fragments of any of the polypeptides described in Table 5 or Table 6.

[0041] The nucleic acid may include a polynucleotide sequence encoding the corresponding enzyme in Table 5 or Table 6, or a substantially homologous variant or fragment of any of the polynucleotides.

[0042] Regarding the QA-RhaT / XylT enzyme in Table 5, as illustrated in the examples below, Qs-3-O-RhaT / XylT is considered a dual rhamnosyl / xylsyl ditransferase.

[0043] Qs_0283850 and DN20529_c0_g2_i8 are both considered to be primarily rhamnosyltransferases, while Qs_0283870 is considered to be primarily xylosyltransferases. However, it should be understood that some degree of cross-activity may exist. For the sake of brevity, all these types of enzymes may be referred to herein as Qs-3-O-RhaT / XylT enzymes or enzymes with QA-RhaT / XylT activity.

[0044] Preferably, as part of the method of the present invention, all three types of enzymes (QA-GlcAT, QA-GalT, and QA-RhaT / XylT) are provided.

[0045] Preferred genes or polypeptides associated with QA-GlcAT, QA-GalT, QA-RhaT / XylT activities for use in the practice of this invention are shown in Table 5 or Table 6 (and Sequence Annex) herein, or substantially homologous variants or fragments of these sequences that have, retain, or encode the necessary biological activities.

[0046] For example, QA-GalT activity can be provided by Qs-3-O-GalT, or alternatively by GmUGT73P2 (which has been shown to have this activity in this paper), or by any of these sequences that are substantially homologous variants or fragments that retain this biological activity.

[0047] In a preferred embodiment, one, two, or three of the polypeptides (i), (ii), or (iii) are selected from the corresponding amino acid sequences listed in Table 5.

[0048] In one implementation, the corresponding polypeptide is selected from a list consisting of:

[0049] (i)QA-GlcAT as shown in SEQ ID: No 2 or 26, or most preferably 2;

[0050] (ii) QA-GalT as shown in SEQ ID: No. 4;

[0051] (iii) QA-RhaT / XylT as shown in SEQ ID: No 6, 28, 30 or 32, with the most preferred option being 6;

[0052] Or any substantially homologous variant or fragment of the stated polypeptide.

[0053] For the sake of brevity, in the context of this invention, and particularly in the methods and uses described herein, the polypeptide or nucleotide sequence in any of Tables 5 and 6 may be referred to herein as “QA-3-O-TriS biosynthetic sequence” or “QA-3-O-TriS sequence”, such as the QA-3-O-TriS gene and the QA-3-O-TriS polypeptide.

[0054] variants

[0055] In addition to using these QA-3-O-TriS genes (and peptides), this invention also covers variants using these genes (and peptides).

[0056] The “variant” QA-3-O-TriS nucleic acid or QA-3-O-TriS polypeptide molecule is homologous or identical to all or part of the QA-3-O-TriS gene or polypeptide sequence discussed in this article.

[0057] The variant peptide possesses the same biological activities as the natural QA-3-O-TriS peptide. The variant nucleic acid encodes the associated variant peptide.

[0058] In this context, the “bioactivity” of the QA-3-O-TriS peptide refers to its ability to catalyze the corresponding reactions shown in Figure 3 and described above, and / or the activities listed in the corresponding tables (e.g., Table 5 or Table 6). The relevant bioactivity can be determined based on the in vitro reactions shown in Table 5. Alternatively, they can be determined by the in vivo activity described in the examples (i.e., by introducing multiple heterologous constructs into the host to produce the corresponding products, which can be determined by LC-MS, etc.).

[0059] Variants of Qs-3-O-RhaT / XylT may include those that preferentially catalyze D-xylose (Xylp) or L-rhamnose (Rhap).

[0060] Alignments for assessing homology can be obtained, for example, using Clustal Omega (version 1.2.4 - accessible via https: / / www.ebi.ac.uk; see, for example, Sievers F et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7(1):539).

[0061] Variants of the sequences disclosed herein preferably have at least 50%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, or 80% identity with a reference polypeptide or polynucleotide sequence, and most preferably at least about 90%, 95%, 96%, 97%, 98%, or 99% identity. Such variants may be referred to herein as “substantially homologous.” Preferred variants have at least 80% identity.

[0062] Preferred variants may be:

[0063] (i) Naturally occurring nucleic acids, such as alleles (which will include polymorphisms or mutations at one or more bases) or pseudoallelic genes (possibly located at loci closely linked to the QA-3-O-TriS gene of the present invention). Also included are paralogous genes, isoglosses, or other homologous genes belonging to the same family as the QA-3-O-TriS gene of the present invention, such as those sharing a clade or subclade. Also included are orthologs or homologs from other plant species.

[0064] As discussed below, homology can be at the nucleotide sequence and / or amino acid sequence level.

[0065] (ii) Artificial nucleic acids, which can be prepared by a person skilled in the art according to this disclosure. For example, such derivatives can be prepared by site-directed or random mutagenesis or by direct synthesis. Preferably, the variant nucleic acid is produced 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 the QA-3-O-TriS gene of the present invention.

[0066] It also includes nucleic acids that correspond to the above nucleic acids but extend to the 3' or 5' end.

[0067] The term “QA-3-O-TriS variant nucleic acid” as used herein encompasses all these possibilities. When used in the context of peptides or proteins, it refers to the expression product encoded by the variant nucleic acid.

[0068] In each case, the preferred QA-3-O-TriS biosynthetic modified nucleic acid is any one of SEQ ID No. 1, 3, 5 or 25, 27, 29 or 31 or a substantially homologous variant thereof. In one embodiment (this aspect or other aspects of the invention relating to these sequences), they are any one of SEQ ID No. 1, 3, 5 or a substantially homologous variant thereof.

[0069] Preferred QA-3-O-TriS biosynthetic modified peptides are any one of SEQ ID No. 2, 4, 6, 26, 28, 30, or 32, or substantially homologous variants thereof. In one embodiment (this aspect or other aspects of the invention relating to these sequences), they are any one of SEQ ID No. 2, 4, or 6, or substantially homologous variants thereof.

[0070] Other preferred QA-3-O-TriS biosynthetic modified nucleic acids used in this invention are SEQ ID No. 19 or a substantially homologous variant or fragment thereof. Other preferred QA-3-O-TriS biosynthetic modified polypeptides are polypeptides encoded by any of these sequences or variants or fragments, such as SEQ ID No. 20.

[0071] Gene supplementation

[0072] In embodiments of the present invention, in addition to the QA-3-O-TriS gene and variant nucleic acid described herein, other genes that may affect the QA-3-O-TriS production throughput may preferably be introduced.

[0073] As illustrated in the previously submitted, unpublished PCT / EP2018 / 086430 (later published as WO 2019 / 122259), the core glycoside ligand (i.e., QA) of QS-21 is a simple triterpenoid derivative of β-amyrin, which is synthesized by cyclizing the universal linear precursor 2,3-oxidized squalene (OS) with oxidized squalene cyclase (OSC).

[0074] The β-amyrin scaffold is further oxidized at positions C-16α, C-23, and C-28 by alcohols, aldehydes, and carboxylic acids, respectively, to form saponin acid.

[0075] Therefore, the biosynthesis of QA from OS involves at least four distinct enzymatic steps. The enzymes involved include:

[0076] • Oxidized squalene cyclase;

[0077] • An enzyme capable of oxidizing β-amyrin or its oxidized derivative at the C-28 position to carboxylic acid;

[0078] • An enzyme that can oxidize β-amyrin or its oxidized derivatives (such as oleanolic acid) at the C-16α position to alcohols;

[0079] • An enzyme that can oxidize β-amyrin or its oxidized derivatives (such as echinocolic acid) at the C-23 position to aldehydes.

[0080] For example:

[0081] (i) β-amyrin synthase (bAS) for cyclizing 2,3-oxidized squalene (OS) into triterpenes;

[0082] (ii) An enzyme capable of oxidizing β-amyrin or its oxidized derivative at the C-28 position to carboxylic acid (“C-28 oxidase”);

[0083] (iii) An enzyme capable of oxidizing β-amyrin or its oxidized derivative at the C-16α position to an alcohol (“C-16α oxidase”); and

[0084] (iv) An enzyme capable of oxidizing β-amyrin or its oxidized derivative at the C-23 position to an aldehyde (“C-23 oxidase”).

[0085] For the sake of brevity, these enzymes can be referred to as “bAS”, “C-28 oxidase”, “C-16α oxidase” and “C-23 oxidase” respectively in this article.

[0086] For the sake of brevity, these enzymes can be collectively referred to as "QA peptides" in this article.

[0087] This invention can be advantageously combined with these QA peptides and encoding nucleic acids.

[0088] Therefore, it should be understood that the combined use of the above-mentioned QA polypeptide and gene with the QA-3-O-TriS polypeptide and gene is explicitly contemplated in any aspect of the materials or methods for the biosynthesis of QA-3-O-TriS in this invention.

[0089] In one implementation scheme:

[0090] C-28 oxidase is CYP716

[0091] C-16α is either CYP716 or CYP87.

[0092] C-23 oxidase is CYP714, CYP72, or CYP94.

[0093] Preferred QA genes or QA peptides, or their bioactive fragments or variants, are shown in Table 8. Variants may be homologs, alleles, or artificial derivatives discussed in relation to the QA-3-O-TriS gene or peptide described above.

[0094] For example, the QA peptide can be any one or more of the following (preferably all of them):

[0095] (i) β-amyrin synthase (bAS) shown in SEQ ID: No 12;

[0096] (ii) C-28 oxidase as shown in SEQ ID: No 14;

[0097] (iii) C-16α oxidase as shown in SEQ ID: No 16;

[0098] (iv) C-23 oxidase as shown in SEQ ID: No 18;

[0099] Or any substantially homologous variant or fragment of the stated polypeptide.

[0100] Mevalonic acid (MVA) is an important intermediate in the synthesis of triterpenoids. Therefore, it may be necessary to express the rate-limiting gene of the MVA pathway into the host to maximize the yield of QA.

[0101] HMG-CoA reductase (HMGR) is considered the rate-limiting enzyme in the MVA pathway.

[0102] It has been shown that transient expression of the recombinant feedback-insensitive truncated form of HMGR (tHMGR) in N. benthamiana increases the content of triterpenoids (β-amyrin)

[19] .

[0103] Therefore, one embodiment of the present invention includes the use of a heterologous HMGR (e.g., a feedback-insensitive HMGR) and the QA-3-O-TriS gene described herein. Examples of HMGR coding sequences or polypeptide sequences include SEQ ID Nos. 7 to 10 or variants or fragments thereof. Variants may be homologs, alleles, or artificial derivatives discussed in relation to the QA-3-O-TriS gene or polypeptide described above. For example, a host-native HMGR, such as yeast HMGR in a yeast host, may be preferred. HMGR genes are known in the art and may be appropriately selected according to this disclosure.

[0104] ***

[0105] Squalene synthase (SQS) has been reported as a possible rate-limiting step.

[19]

[0106] Therefore, one embodiment of the present invention includes the use of heterologous SQS, as well as the QA-3-O-TriS gene described herein and optional HMGR.

[0107] Examples of SQS coding sequences or polypeptide sequences include SEQ ID Nos. 21 to 22 or variants or fragments thereof. Variants may be homologs, alleles, or artificial derivatives discussed in relation to the QA-3-O-TriS gene or polypeptide described above. For example, a host-native SQS may be preferred, such as yeast SQS in a yeast host. The SQS gene is known in the art and may be appropriately selected according to this disclosure.

[0108] When using certain hosts (e.g., yeast), it may be necessary to introduce additional genes to increase QA production throughput. Examples may include one or more plant cytochrome P450 reductases (CPRs) as redox chaperones for the introduced P450. Therefore, one embodiment of the invention includes the use of a heterologous cytochrome P450 reductase (such as AtATR2, i.e., Arabidopsis thaliana cytochrome P450 reductase 2) and the QA polypeptide and QA-3-O-TriS gene described herein. Examples of AtATR2 coding sequences or polypeptide sequences include SEQ ID Nos. 23 to 24 or variants or fragments thereof. Variants may be homologs, alleles, or artificial derivatives discussed in connection with the QA-3-O-TriS gene or polypeptide described above.

[0109] Therefore, in one embodiment, the nucleic acid used in this invention further encodes one or more of the following polypeptides:

[0110] (i) HMG-CoA reductase (HMGR);

[0111] (ii) Squalene synthase (SQS)

[0112] The HMGR or SQS therein is optionally selected from the corresponding polypeptide in Table 7 or a substantially homologous variant or fragment of any of the polypeptides, or encoded by the corresponding polynucleotide in Table 7 or a substantially homologous variant or fragment of any of the polynucleotides.

[0113] As will be understood by those skilled in the art based on this disclosure, additional genes may be used in the practice of this invention to provide additional activity and / or improve expression or activity. These include those expressing cofactors or accessory proteins or other factors.

[0114] For the sake of brevity, unless the context otherwise requires, any of these nucleic acid sequences (“the QA-3-O-TriS gene of the present invention”, plus other genes affecting QA-3-O-TriS synthesis, or secondary modifications of QA-3-O-TriS) may be referred to herein as “QA-3-O-TriS biosynthetic modified nucleic acid”. Similarly, the encoded polypeptide may be referred to herein as “QA-3-O-TriS biosynthetic modified polypeptide”.

[0115] It should be understood that, where these general terms are used in connection with any aspect or implementation, the meaning or disclosure therein shall be deemed to be applied individually to any of these sequences with the necessary modifications.

[0116] carrier

[0117] As one aspect of the present invention, a method for co-osmosis using multiple Agrobacterium tumefaciens strains is disclosed, each Agrobacterium tumefaciens strain carrying one or more QA-3-O-TriS nucleic acids discussed above for co-expression in the biosynthetic pathways discussed above.

[0118] In some implementations, at least two or three different Agrobacterium tumefaciens strains are co-permeated, for example, each Agrobacterium tumefaciens strain carries QA-3-O-TriS nucleic acid.

[0119] The gene may have originated from a transient expression vector.

[0120] A preferred expression system utilizes the so-called "Hyper-Translatable" Cowpea Mosaic Virus (CPMV-HT) system described in WO 2009 / 087391, the disclosure of which is specifically incorporated herein to support embodiments using the CPMV-HT system (e.g., vectors based on pEAQ-HT expression plasmids).

[0121] Therefore, the carrier (typically a binary carrier) used in this invention will generally include an expression cassette, which comprises:

[0122] (i) a promoter operatively connected to

[0123] (ii) an enhancer sequence of an RNA-2 genome segment derived from a dimorphic RNA virus, wherein the target start site in the RNA-2 genome segment has been mutated;

[0124] (iii) The QA-3-O-TriS nucleic acid sequence as described above;

[0125] (iv) Terminate the subsequence; and optionally

[0126] (v) The 3'UTR located upstream of the termination subsequence.

[0127] Further examples of carriers and expression systems that can be used in the practice of this invention are described in more detail below.

[0128] Host

[0129] In some aspects of the invention, a host phenotype that cannot efficiently biosynthesize QA-3-O-TriS from OS can be converted into a host phenotype that can perform said QA-3-O-TriS biosynthesis, so that QA-3-O-TriS can be recovered therefrom or used in vivo for the synthesis of downstream products.

[0130] As illustrated above, QA biosynthesis can also be engineered into plants based on the disclosure of the previously filed, unpublished PCT / EP2018 / 086430 (later published as WO 2019 / 122259). Since QA precursors (2,3-squalene oxide) are ubiquitous in higher plants due to their role in sterol biosynthesis, this invention has broad applicability to plant hosts. As discussed herein, additional activities can be used when practicing this invention in microorganisms.

[0131] Examples of hosts include plants (such as Nicotiana benthamiana) and microorganisms (such as yeast). These are discussed in more detail below.

[0132] The present invention may include transforming a host with a heterologous nucleic acid as described above by introducing QA-3-O-TriS nucleic acid into a host cell via a vector and inducing or allowing recombination between the vector and the host cell genome to introduce the nucleic acid according to the invention into the genome.

[0133] In another aspect of the invention, a host cell transformed with a heterologous nucleic acid is provided, the heterologous nucleic acid comprising a plurality of nucleotide sequences, each of the plurality of nucleotide sequences encoding a polypeptide having, in combination, the QA-3-O-TriS biosynthetic activity.

[0134] The expression of the nucleic acid thereon confers on the transformed host the ability to perform QA-3-O-TriS biosynthesis from OS, or enhances the ability in the host.

[0135] This invention further covers host cells, particularly plant or microbial cells, transformed by the aforementioned nucleic acids or vectors (e.g., containing QA-3-O-TriS biosynthetic modified nucleotide sequences). In transgenic host cells (i.e., transgenic with respect to the nucleic acids in question), the transgene can be incorporated into the genome via an extragenomic vector or preferably stably. More than one heteronucleotide sequence may be present in each haploid genome.

[0136] The methods and materials described herein are particularly useful for producing stable crop plants that accumulate QA-3-O-TriS. Examples of plants include intercropping crops such as sunflower, potato, canola, bean sprouts, field pea, flax, safflower, buckwheat, cotton, corn, soybean, and sugar beet. Major crop plants such as maize, wheat, rapeseed, and rice can also be preferred hosts.

[0137] A plant comprising plant cells according to the invention is also provided.

[0138] Product production

[0139] The methods described above can be used to generate glycosylated QAs, such as QA-3-O-TriS, in heterologous hosts. Glycosylated QAs (such as QA-3-O-TriS) are typically not naturally present in the species to which they are introduced.

[0140] Glycosylated QA (such as QA-3-O-TriS) can be isolated from the plants or methods of this invention and commercially developed.

[0141] The above-described method may constitute part of, or possibly be a step in, a method for producing downstream products (such as QS-21) in a host. The method may include the steps of: culturing the host (in the case of a microorganism) or causing the host to grow (in the case of a plant), then harvesting the host and purifying the glycosylated QA (such as QA-3-O-TriS) or its downstream products or derivatives (e.g., QS-21). The product thus produced constitutes another aspect of the invention. The uses of QS-21 are as described above.

[0142] Alternatively, glycosylated QA (such as QA-3-O-TriS) can be recovered to allow for further chemical synthesis of downstream compounds.

[0143] The novel gene of this invention

[0144] To support this invention, the inventors have newly characterized or identified sequences from soap trees that are believed to be involved in the synthesis of glycosylated compounds (see SEQ.ID: No 1-6 and 25-32).

[0145] In one embodiment (this aspect or other aspects of the invention relating to these sequences), the sequences are selected from any one of SEQ ID No. 1-6.

[0146] In a preferred embodiment, the method of the present invention will include using one or more of these newly characterized QA-3-O-TriS nucleic acids of the present invention (e.g., one, two or three such QA-3-O-TriS nucleic acids), optionally in combination with manipulation of other genes known in the art that affect QA biosynthesis.

[0147] These newly characterized QA-3-O-TriS sequences (SEQ.ID: No. 1-6 and 25-32) from soap trees constitute aspects of the present invention in themselves, as do the derived variants and materials of these sequences and the methods of using them.

[0148] Some aspects and embodiments of the invention will now be described in more detail. Detailed Implementation

[0149] The inventors used various genomic and transcriptomic approaches related to soapberry to begin elucidating the biosynthetic pathway associated with the glycosylation of QA. Functional characterization of candidate genes by transient expression in *Nicotiana benthamiana* led to the identification of three enzymes from soapberry that, together, can biosynthesize QA-3-O-TriS from QA.

[0150] In various embodiments, the present invention provides a method for manipulating the total level of QA in glycosylation in host cells (such as microorganisms or plants).

[0151] In one aspect of the invention, the above-mentioned QA-3-O-TriS modified nucleic acid is in a recombinant form and preferably in a reproducible vector.

[0152] "Vector" is defined in particular as any plasmid, granule, phage, or Agrobacterium binary vector in double-stranded or single-stranded linear or circular form, which may or may not be self-transmittable or mobile, and may transform prokaryotic or eukaryotic hosts by integration into the cell genome or may exist outside the chromosome (e.g., autonomously replicating plasmids with origin of replication).

[0153] As is well known to those skilled in the art, a “binary vector” system comprises: (a) a boundary sequence that allows the transfer of a desired nucleotide sequence into the plant cell genome; and (b) the desired nucleotide sequence itself, which typically contains (i) an expression cassette of a plant active promoter appropriately and operatively linked to (ii) a target sequence and / or an enhancer. The desired nucleotide sequence is located between the boundary sequences and is capable of insertion into the plant genome under appropriate conditions. Binary vector systems typically require additional sequences (derived from *Agrobacterium tumefaciens*) for integration. This is typically achieved using so-called “agro-infiltration,” which utilizes *Agrobacterium*-mediated transient transformation. In short, this technique is based on the property of *Agrobacterium tumefaciens* to transfer a portion of its DNA (“T-DNA”) into a host cell, where the T-DNA may integrate into the nuclear DNA. The T-DNA is defined by left and right boundary sequences of approximately 21-23 nucleotides in length. Infiltration can be achieved, for example, by a syringe (in a leaf) or by vacuum (the entire plant). In this invention, the boundary sequence will typically be included around the desired nucleotide sequence (T-DNA), wherein one or more vectors are introduced into the plant material via Agrobacterium infiltration.

[0154] Generally, those skilled in the art are well capable of constructing vectors and design schemes for recombinant gene expression (e.g., for expressing heterologous nucleic acids in a host or one or more host cells). Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other suitable sequences. For further details, see, for example, *Molecular Cloning: a Laboratory Manual: 2nd Edition*, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press, or *Current Protocols in Molecular Biology*, 2nd Edition, edited by Ausubel et al., John Wiley & Sons, 1992.

[0155] Specifically, this includes shuttle vectors, which are DNA carriers that are naturally or intentionally designed to replicate in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotes (e.g., higher plants, bryophytes, yeast or fungal cells).

[0156] Vectors containing nucleic acids according to the invention do not need to include promoters or other regulatory sequences, especially when the vector is used to introduce the nucleic acid into a cell for recombination into the genome.

[0157] Preferably, the nucleic acid in the vector is transcribed in a host cell (such as a microbial cell, e.g., yeast or bacterial cell) or operably linked to a suitable promoter or other regulatory element. The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory element (optionally combined with a heterologous enhancer, such as the 35S enhancer discussed in the examples below). The advantage of using a natural promoter is that pleiotropic responses can be avoided. In the case of cDNA, it may be expressed in a host cell under the control of a suitable promoter or other regulatory element.

[0158] A “promoter” refers to a nucleotide sequence from which transcription is initiated operatively to be linked to downstream (i.e., the 3' direction of the sense strand of a double-stranded DNA) DNA.

[0159] "Operationally ligated" means that the DNA is ligated as part of the same nucleic acid molecule, properly positioned and oriented to initiate transcription from the promoter. DNA that is operatively ligated to the promoter is "under the regulation of transcription initiation" of the promoter.

[0160] In a preferred embodiment, the promoter is an inductive promoter.

[0161] The term "inducible" applied to promoters is well known to those skilled in the art. Essentially, expression under the control of an inducible promoter is "turned on" or increased in response to an applied stimulus. The nature of the stimulus varies from promoter to promoter. Some inducible promoters elicit little or no expression (or no expression) in the absence of a suitable stimulus. Other inducible promoters elicit detectable constitutive expression in the absence of a stimulus. Regardless of the expression level in the absence of a stimulus, the expression of any inducible promoter will increase in the presence of a suitable stimulus.

[0162] Therefore, the nucleic acid according to the invention can be placed under the control of an externally inducible gene promoter, so that expression (expression of a heterologous sequence) is under the user's control. The advantage of introducing a heterologous gene into plant cells (especially when the cell is contained within a plant) is the ability to place gene expression under the control of a selected promoter, allowing for the influence of gene expression according to preference, thus enabling QA-3-O-TriS biosynthesis. Furthermore, mutants and derivatives of the wild-type gene (e.g., having higher or lower activity than the wild-type) can be used to replace the endogenous gene.

[0163] Therefore, this aspect of the invention provides a gene construct, preferably a reproducible vector, comprising a promoter (optionally an inducible promoter) operatively linked to a nucleotide sequence provided herein, such as a QA-3-O-TriS biosynthetic modification gene, most preferably one of the QA-3-O-TriS nucleic acids described herein or a derivative thereof.

[0164] In this context, nucleic acid constructs used as plant vectors are of particular interest. Specific procedures and vectors previously used with widespread success in plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (edited by CroyRRD), Oxford, BIOS Scientific Publishers, pp. 121–148). Suitable vectors may include plant virus-derived vectors (see, for example, EP-A-194809).

[0165] Preferably, the vector of the present invention for use in plants contains a boundary sequence that allows expression cassette transfer and integration 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, DA, LW Harris-Haller, et al. (1995). "Complete Sequence of the binary vector Bin 19." Plant Molecular Biology 27:405-409).

[0166] Suitable promoters that function in plants include cauliflower mosaic virus 35S (CaMV 35S). Other examples are disclosed on page 120 of Lindsey and Jones (1989), "Plant Biotechnology in Agriculture" (published by OU Press, Milton Keynes, UK). Promoters can be selected to include one or more sequence motifs or elements that confer developmental and / or tissue-specific regulatory control over expression. Inducible plant promoters include the ethanol-inducible promoter described by Caddick et al. (1998), Nature Biotechnology 16:177-180.

[0167] If desired, selective genetic markers can be included in the construct, such as those that confer selective phenotypes (e.g., resistance to antibiotics or herbicides such as kanamycin, hygromycin, glufosinate, chlorsulfuron, methotrexate, gentamicin, spectinomycin, imidazolinone, and glyphosate). Positive selection systems (such as those described by Haldrup et al., 1998, Plantmolecular Biology 37, 287-296) can be used to create antibiotic-independent constructs.

[0168] As explained above, the preferred vector is the 'CPMV-HT' vector as described in WO 2009 / 087391. The following examples demonstrate the use of these pEAQ-HT expression plasmids.

[0169] These carriers (typically binary carriers) used in this invention will generally include an expression cassette, which comprises:

[0170] (i) a promoter operatively connected to

[0171] (ii) Enhancer sequences of RNA-2 genome segments derived from dimorphic RNA viruses, wherein the target start site in the RNA-2 genome segment has been mutated;

[0172] (iii) The QA-3-O-TriS nucleic acid sequence as described above;

[0173] (iv) Terminate the subsequence; and optionally

[0174] (v) The 3'UTR located upstream of the termination subsequence.

[0175] As used herein, "enhancer" sequences (or enhancer elements) are sequences derived from (or homologous to) RNA-2 genomic segments of dimorphic RNA viruses (such as cowpea mosaic virus), in which the target start site has been mutated. Such sequences can enhance downstream expression of the heterologous ORF to which they are attached. Without limitation, it is thought that the presence of such sequences in transcribed RNA can enhance the translation of the heterologous ORF to which they are attached.

[0176] As referred to herein, the “target initiation site” is the initiation site (start codon) in the wild-type RNA-2 genome segment of a dimorphic virus (e.g., cowpea mosaic virus). The enhancer sequence discussed is derived from the initiation site, which serves as the initiation site for generating (translating) the longer of the two carboxyl-terminated proteins encoded by the wild-type RNA-2 genome segment.

[0177] Typically, the RNA virus would be the cowpea mosaic virus described above.

[0178] The most preferred vector is the pEAQ vector of WO 2009 / 087391, which allows direct cloning of a form located on T-DNA via a multiple adapter between the 5' leader sequence and the 3' UTR of the expression cassette containing the translation enhancer of the present invention, which also contains a gene silencing repressor and an NPTII cassette.

[0179] The presence of gene silencing repressors in such gene expression systems is preferred, but not essential. Gene silencing repressors are known in the art and described in WO / 2007 / 135480. They include HcPro from Potato Virus Y, He-Pro from TEV, P19 from TBSV, rgsCam, B2 protein from FHV, small coat protein from CPMV, and coat protein from TCV. When producing stable transgenic plants, the preferred repressor is the P19 repressor incorporated with the R43W mutation.

[0180] The present invention also provides a method comprising introducing such construct into plant cells or microbial (e.g., bacteria, yeast, or fungi) cells and / or inducing expression of the construct within plant cells by applying a suitable stimulus (e.g., an effective exogenous inducer).

[0181] As a substitute for microorganisms, cell suspension cultures of engineered plant species that produce glycosylated QA (including the moss Physcomitrella patens) can be cultured in fermenters (see, for example, Grotewold et al. (Engineering Secondary Metabolites in Maize Cells by Ectopic Expression of Transcription Factors, Plant Cell, 10, 721-740, 1998)).

[0182] In another aspect of the invention, host cells, particularly plant or microbial cells, containing heterologous constructs according to the invention are disclosed.

[0183] The above discussion of host cells related to the re-establishment of QA-3-O-TriS biosynthesis in heterologous organisms is applicable here with necessary modifications.

[0184] Therefore, another aspect of the present invention provides a method for transforming plant cells, the method comprising introducing the construct as described above into plant cells and inducing or allowing recombination between the vector and the plant cell genome to introduce the nucleic acid invention according to the present invention into the genome.

[0185] This invention further covers host cells, particularly plant or microbial cells, transformed with nucleic acids or vectors according to the invention (e.g., containing QA-3-O-TriS biosynthetic modified nucleotide sequences). In transgenic plant cells (i.e., transgenic with respect to the nucleic acid in question), the transgene can be incorporated into the genome on an extragenomic vector or preferably stably. More than one heteronucleotide sequence may be present in each haploid genome.

[0186] Yeast has been widely used as a host for the production of triterpenes and is therefore likely well suited for the biosynthesis of QA followed by QA-3-O-TriS.

[0187] Therefore, in one implementation, the host is yeast. For such a host, it may be necessary to introduce additional genes to increase the QA production flux as described above, and thus increase the QA-3-O-TriS production flux. Examples may include one or more plant cytochrome P450 reductases (CPRs) as redox chaperones for the introduced P450, and HMGR. Similarly, it may be necessary to introduce additional genes to contribute other elements of QA or to improve the QA-3-O-TriS pathway. These may include enzymes that provide UDP-glucose donors, etc. (see, for example, 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 UDP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae”. FEBS J. 273(12):2645-57). According to this disclosure, those skilled in the art can provide such auxiliary activities as needed.

[0188] Plants comprising the plant cells transformed as described above constitute another aspect of the present invention.

[0189] If needed, as is the standard practice in this field, plants can regenerate, for example, from single cells, callus, or leaf discs after transformation of plant cells. Almost any plant can regenerate completely from its cells, tissues, and organs. Available techniques are reviewed in Vasil et al., *Cell Culture and Somatic Cell Genetics of Plants*, Volumes I, II, and III, *Laboratory Procedures and Their Applications*, Academic Press, 1984, and Weissbach and Weissbach, *Methods for Plant Molecular Biology*, Academic Press, 1989.

[0190] In addition to regenerated plants, the present invention includes all of the following: clones, seeds, self-pollinated or hybrid offspring and descendants (e.g., F1 and F2 offspring) of such plants. The invention also provides plant propagules from such plants, i.e., any part, sexual or asexual, that can be used in reproduction or propagation, including cuttings, seeds, etc. In all cases, these plants or parts comprise the aforementioned plant cells or heterologous QA-3-O-TriS biosynthetic modified DNA (e.g., as introduced into ancestral plants).

[0191] It also provides any part of these plants (e.g., leaves, stems, dried or ground products, edible parts, etc.), which in all cases includes the aforementioned plant cells or heterologous QA-3-O-TriS biosynthesized modified DNA.

[0192] The present invention also covers expression products of any disclosed QA-3-O-TriS biosynthetic modified nucleic acid sequences, and therefore methods for preparing said expression products (which may be in suitable host cells) by expression from the encoding nucleic acid under suitable conditions.

[0193] As described below, the plant background (such as those mentioned above) can be natural or genetically modified, for example, for one or more genes that are involved in or otherwise affect the phenotype or trait of glycosylated QA (such as QA-3-O-TriS).

[0194] When modifying the host phenotype, the QA-3-O-TriS nucleic acid described herein can be used in combination with any other gene (such as a transgene or its modification that affects the rate or yield of QA-3-O-TriS) or any other phenotypic trait or desired characteristic.

[0195] By using combinations of genes, plants or microorganisms (such as bacteria, yeast, or fungi) can be customized to enhance the production of desired precursors or reduce unwanted metabolism.

[0196] As an alternative, it may be necessary to downregulate genes in the host, for example, to reduce unwanted metabolism or flux that could affect QA-3-O-TriS production.

[0197] Such downsampling can be achieved using methods known in the art, such as the use of antisense techniques.

[0198] When using antisense genes or portions of gene sequences to downregulate gene expression, the nucleotide sequence is placed under the control of an "inverted" promoter, causing transcription to produce RNA complementary to the 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 techniques are also reviewed in Bourque, (1995), Plant Science 105, 125-149 and Flavell, (1994) PNAS USA 91, 3490-3496.

[0199] An alternative approach to antisense is to use a copy of the target gene, either fully or partially, inserted in the meaningful direction (i.e., the same direction as the target gene) to reduce the expression of the target gene through 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 gene silencing or co-suppression techniques can be found in: WO 95 / 34668 (Biosource); Angell & Baulcombe (1997) The EMBO Journal 16, 12: 3675-3684; and Voinnet & Baulcombe (1997) Nature 389: p. 553.

[0200] Double-stranded RNA (dsRNA) has been found to be more effective at gene silencing than single sense or antisense strands (Fire A. et al., Nature, Vol. 391, (1998)). dsRNA-mediated silencing is gene-specific and is often referred to as 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).

[0201] RNA interference is a two-step approach. First, dsRNA is cleaved within the cell to produce a short interfering RNA (siRNA) of approximately 21–23 nt in length, with a 5' phosphate terminus and a short 3' overhang (approximately 2 nt). The siRNA targets the corresponding mRNA sequence specifically designed for disruption (Zamore PDNature Structural Biology, 8, 9, 746–750, (2001)).

[0202] Another method known in the art for downregulating target sequences is the use of “microRNAs” (miRNAs), for example, as described by Schwab et al. 2006, Plant Cell 18, 1121-1133. This technique employs artificial miRNAs that can be encoded by stem-loop precursors incorporated with suitable oligonucleotide sequences, which can be generated according to the disclosure herein using well-defined rules.

[0203] Therefore, in one aspect, the present invention provides a method for influencing or affecting the biosynthesis of glycosylated QA in a host, said method comprising any of the following steps:

[0204] (i) Inducing or allowing transcription from nucleic acids containing a complementary sequence of QA-3-O-TriS nucleotide sequence, for example, to reduce the activity of the respective encoded polypeptides via antisense mechanisms;

[0205] (ii) Inducing or allowing transcription from the nucleic acid encoding the stem-loop precursor, for example by reducing the activity of the respective encoded polypeptide via a miRNA mechanism, said stem-loop precursor comprising 20-25 nucleotides of the QA-3-O-TriS nucleotide sequence (optionally including one or more mismatches);

[0206] (iii) Inducing or allowing transcription from a nucleic acid encoding a double-stranded RNA, for example by reducing the activity of the respective encoded polypeptide via an siRNA mechanism, wherein the double-stranded RNA corresponds to 20-25 nucleotides of the QA-3-O-TriS nucleotide sequence (optionally including one or more mismatches).

[0207] The methods of the present invention include the in vitro and in vivo production or manipulation of one or more glycosylated QAs. For example, QA-3-O-TriS polypeptides can be used in fermentation via expression in microorganisms such as E. coli, yeast, and filamentous fungi. In one embodiment, one or more novel characterized QA-3-O-TriS sequences of the present invention can be used in these organisms in conjunction with one or more other biosynthetic genes.

[0208] The above describes in vivo methods extensively, and they typically involve steps that induce or allow transcription of a recombinant nucleic acid molecule encoding the QA-3-O-TriS polypeptide, followed by translation from it.

[0209] In other aspects of the invention, the QA-3-O-TriS polypeptide (enzyme) can be used in vitro, for example, in isolated, purified, or semi-purified form. Optionally, they can be expression products of recombinant nucleic acid molecules.

[0210] The newly characterized sequence from soapberry

[0211] As described above, in support of the present invention, the inventors have identified genes from the soapberry tree that are believed to encode polypeptides that affect the biosynthesis of QA-3-O-TriS (see SEQ.ID: No 1-6 and 25-32 in Table 5).

[0212] Therefore, the newly characterized QA-3-O-TriS biosynthetic gene from soapberry itself constitutes an aspect of the present invention.

[0213] In another aspect of the invention, a nucleic acid is disclosed, which is a variant of the QA-3-O-TriS nucleic acid derived from the soap tree discussed above.

[0214] Similar to the natural genes discussed herein, such variants can be used to alter the content of glycosylated QA (e.g., QA-3-O-TriS) in plants, as assessed by the methods disclosed herein. For example, the variant nucleic acid may include a sequence encoding a variant QA-3-O-TriS polypeptide having the relevant biological activities of the natural QA-3-O-TriS polypeptide, as discussed above. Examples include variants of any one of SEQ ID Nos. 2, 4, 6, 26, 28, 30, or 32.

[0215] derivative

[0216] This article describes a method for producing nucleic acid derivatives, the method comprising the step of modifying any of the present invention's QA-3-O-TriS genes disclosed above (particularly the QA-3-O-TriS sequence from soapberry).

[0217] For a variety of reasons, modifications may be necessary. For example, they may introduce or remove restriction endonuclease sites or alter codon usage. This may be particularly desirable in cases where the QA-3-O-TriS gene is to be expressed in an alternative host (e.g., a microbial host, such as yeast). Methods for codon-optimized genes for this purpose are known in the art (see, for example, Elena, Claudia et al., "Expression of codon-optimized genes in microbial systems: current industrial applications and perspectives." Frontiers in Microbiology 5 (2014)). Therefore, the sequences described herein that include codon modifications to maximize yeast expression represent specific embodiments of the invention.

[0218] Alternatively, alterations to the sequence can produce derivatives by adding, inserting, deleting, or substituting one or more nucleotides in a nucleic acid, resulting in the addition, insertion, deletion, or substitution of one or more amino acids in the encoded polypeptide.

[0219] This modification can alter the sites required for post-translational modification, such as cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide used for phosphorylation, etc. Leader sequences or other targeting sequences (e.g., membrane or Golgi localization sequences) can be added to the expressed protein to determine its post-expression location (if it is desired to isolate it from the microbial system).

[0220] Other desirable mutations can be random or site-directed mutagenesis to alter the activity (e.g., specificity) or stability of the encoded peptide. Changes can be made through conserved variations, i.e., substituting one hydrophobic residue (such as isoleucine, valine, leucine, or methionine) for another, or substituting one polar residue for another, such as substituting arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a peptide through conserved substitution may not significantly change its activity because the side chains of the inserted amino acids may form bonds and linkages similar to those of the substituted amino acids. This is true even if the substitution is located in a region critical to determining the peptide's conformation. Variants with non-conserved substitutions are also included. As is well known to those skilled in the art, substituting regions of a peptide whose conformation is not critical to its activity may not significantly affect its activity because the substitution does not drastically alter the peptide's three-dimensional structure. In regions critical to determining the peptide's conformation or activity, such changes can confer advantageous properties to the peptide. In fact, changes such as those described above can endow peptides with slightly advantageous properties, such as altered stability or specificity.

[0221] Excerpt

[0222] The present invention can utilize gene fragments encoding the QA-3-O-TriS polypeptide disclosed above, particularly the QA-3-O-TriS sequence from soap trees.

[0223] Therefore, this invention provides fragments of the full-length QA-3-O-TriS polypeptide disclosed herein, and in particular the generation and use of its active portion. The “active portion” of a polypeptide refers to a peptide smaller than the full-length polypeptide but retaining its essential biological activity (e.g., activity related to the glycosylation of QA) (see, for example, Table 5).

[0224] The term "fragment" of a polypeptide refers to a segment of amino acid residues having at least about five to seven consecutive amino acids, often at least about seven to nine consecutive amino acids, typically at least about nine to 13 consecutive amino acids, and most preferably at least about 20 to 30 or more consecutive amino acids. A polypeptide fragment may include one or more epitopes that can be used to generate antibodies against any portion of the amino acid sequence disclosed herein. Preferred epitopes are those that antibodies can specifically bind to, which can be considered to bind the polypeptides or fragments thereof of the present invention with at least about 1000 times the affinity of other polypeptides.

[0225] For the sake of brevity, these QA-3-O-TriS sequences or variants (e.g., derivatives, such as fragments thereof) from soap trees may be referred to as “Qs QA-3-O-TriS sequences (or nucleic acids, or polypeptides)”. These Qs QA-3-O-TriS polypeptides, along with the nucleic acids encoding them, constitute an aspect of this invention.

[0226] It should be understood that, in the context of the general use of this term, it also applies individually to any one of these sequences.

[0227] Therefore, in one aspect of the invention, isolated nucleic acids encoding any of these polypeptides (2, 4, 6, 26, 28, 30, or 32) are disclosed. Preferably, the nucleic acid may have a sequence of 1, 3, 5, 25, 27, 29, or 31. Other nucleic acids of the invention include nucleic acids degenerately equivalent to these nucleic acids or homologous variants (e.g., derivatives) of these nucleic acids.

[0228] A further aspect of the invention includes isolated nucleic acids comprising sequences complementary to any sequences discussed below.

[0229] The use of the QA-3-O-TriS sequence in vitro or in vivo for catalyzing its respective biological activities (QA glycosylation, for example, as described in Figure 3 or Figure 9 or Table 5) constitutes another aspect of the invention.

[0230] Therefore, the present invention further provides a method for interfering with or influencing the biosynthesis of glycosylated QA (e.g., QA-3-O-TriS) in a host such as a plant, the method comprising inducing or allowing transcription of heterologous QsQA-3-O-TriS nucleic acid as discussed above within plant cells. This step may precede an earlier step of introducing the QsQA-3-O-TriS nucleic acid into the cells of a plant or its ancestor.

[0231] Such methods are typically incorporated as part of, or may be a step in, a process for producing glycosylated QA (e.g., QA-3-O-TriS) in a host such as a plant. Preferably, the method will use the QsQA-3-O-TriS polypeptide of the present invention as described above (e.g., in Table 5) or a derivative thereof, or a nucleic acid encoding either of these.

[0232] In another embodiment, an antibody is provided that generates a peptide or polypeptide against the Qs QA-3-O-TriS polypeptide or peptide of the present invention.

[0233] Some aspects of the invention will now be discussed in more detail, as they relate to the heterologous reconstruction of the biosynthetic pathways discussed above.

[0234] The term "nucleic acid" according to the invention may include cDNA, RNA, genomic DNA, and modified nucleic acids or nucleic acid analogs (e.g., peptide nucleic acids). Where a DNA sequence is specified, for example with reference to the accompanying drawings, unless the context otherwise requires, it includes RNA equivalents with U replaced by T (where they are present). Nucleic acids may include more than one type of nucleic acid molecule. Nucleic acid molecules according to the invention may be isolated and / or purified from their natural environment, provided in substantially pure or homogeneous form, or in form containing no or substantially no other nucleic acids from the source species, and in double-stranded or single-stranded form. As used herein, the term "isolated" covers all these possibilities. Nucleic acid molecules may be wholly or partially synthetic. In particular, they may be recombinant, since nucleic acid sequences not found together in nature (operating without contact) have been linked or otherwise artificially combined. Nucleic acids may comprise, consist of, or consist substantially of any sequences discussed below.

[0235] The term "complementary sequence" for nucleic acids as used herein refers to a complementary sequence of a nucleic acid or a complementary sequence of nucleotides contained within a nucleic acid. Optionally, the complementary sequence is full-length compared to a reference nucleotide sequence.

[0236] The term "heterologous" is used extensively herein to indicate that the gene / nucleotide sequence in question (e.g., encoding a QA-3-O-TriS biosynthetic modified polypeptide) has been introduced into the cells of the host or its ancestor using genetic engineering (i.e., through artificial intervention). The heterologous nucleic acid will not be naturally present in the cells of that type, variety, or species. Therefore, the heterologous nucleic acid may comprise or be derived from a coding sequence of a specific type of plant cell or plant species or variety, placed in the environment of plant cells of a different plant type or species or variety. A further possibility is to place the nucleic acid sequence within a cell in which it or a homolog is naturally found, but wherein the nucleic acid sequence is linked and / or adjacent to a nucleic acid not naturally present in the cell or cells of that plant type or species or variety, for example, operatively linked to one or more regulatory sequences (e.g., promoter sequences) for controlling expression.

[0237] In this article, "transformed" means that the nucleotide sequence of a heterologous nucleic acid changes one or more cellular characteristics and thus alters the phenotype, such as the biosynthesis of glycosylated QA (e.g., QA-3-O-TriS). This transformation may be transient or stable.

[0238] "Inability to perform QA-3-O-TriS biosynthesis" means that the host does not, or does not believe, to naturally produce detectable or recyclable levels of QA-3-O-TriS under the host's normal metabolic conditions prior to transformation. After applying this invention, it is able to produce detectable or recyclable levels of QA-3-O-TriS.

[0239] The nucleotide sequence information provided herein can be used to design probes and primers for detection or amplification. The length of oligonucleotides used in detection or PCR can be about 30 nucleotides or less (e.g., 18, 21, or 24). Typically, specific primers are longer than 14 nucleotides. Primers of 16-24 nucleotides in length may be preferred for optimal specificity and cost-effectiveness. Those skilled in the art are highly proficient in primer design for methods such as PCR. If desired, the entire restriction fragment of the gene disclosed herein can be used for detection, and the fragment can be 100 or even 1000 nucleotides in length. If desired, small variations can be introduced into the sequence to produce “common” or “degenerate” primers.

[0240] The probe can be performed using standard DNA blotting techniques. For example, DNA can be extracted from cells and digested with different restriction enzymes. The restriction fragments can then be separated by agarose gel electrophoresis, denatured, and transferred to a nitrocellulose filter. A labeled probe can hybridize with the single-stranded DNA fragment on the filter and identify the binding. The DNA used for the probe can be prepared from RNA preparations derived from cells. The probe can optionally be performed using so-called 'nucleic acid chips' (see Marshall and Hodgson (1998) Nature Biotechnology 16:27-31 review).

[0241] In one embodiment, a variant encoding the QA-3-O-TriS biosynthetic modified polypeptide according to the present invention can be obtained by a method comprising:

[0242] (a) A preparation providing, for example, nucleic acids from plant cells. The test nucleic acid can be obtained from the cell as genomic DNA, cDNA, or RNA, or any mixture thereof, preferably as a library of suitable vectors. If genomic DNA is used, the probe can be used to identify untranscribed regions of a gene (e.g., promoters, etc.), as described below.

[0243] (b) Provide nucleic acid molecules that are probes or primers as discussed above.

[0244] (c) Under conditions where the nucleic acid molecule hybridizes with any of the genes or homologs in the preparation, the nucleic acid in the preparation is contacted with the nucleic acid molecule, and

[0245] (d) Identification of the gene or homolog (if present) by hybridization with the nucleic acid molecule. Binding of the probe to the target nucleic acid (e.g., DNA) can be measured using any of a variety of techniques available to those skilled in the art. For example, the probe may be radioactively, fluorescently, or enzymatically labeled. Other methods that do not use probe labeling include PCR amplification (see below), RNase cutting, and allele-specific oligonucleotide detection. Following identification of successful hybridization is the isolation of the hybridized nucleic acid, which may involve one or more steps of PCR or amplification of the vector in a suitable host.

[0246] Preliminary experiments can be conducted by hybridization under less stringent conditions. For probing, the preferred conditions are those that are stringent enough to provide a simple pattern in which a small number of hybridizations are identified as positive for further investigation.

[0247] For example, hybridization can be performed using a hybridization solution comprising: 5X SSC (where 'SSC' = 0.15M sodium chloride; 0.15M sodium citrate; pH 7), 5X Denhardt reagent, 0.5%–1.0% SDS, 100 μg / ml denatured fragmented salmon sperm DNA, 0.05% sodium pyrophosphate, and up to 50% formamide. Hybridization is performed at 37–42°C for at least six hours. After hybridization, the filter is washed as follows: (1) at room temperature in 2X SSC and 1% SDS for 5 minutes; (2) at room temperature in 2X SSC and 0.1% SDS for 15 minutes; (3) at 37°C in 1X SSC and 1% SDS for 30 minutes–1 hour; (4) at 42–65°C in 1X SSC and 1% SDS for 2 hours, changing the solution every 30 minutes.

[0248] A commonly used formula for calculating the stringent conditions required for hybridization between nucleic acid molecules with specific sequence homology is (Sambrook et al., 1989): T m = 81.5℃ + 16.6Log[Na+] + 0.41(%G+C) - 0.63(%formamide) - 600 / #bp in the double strand

[0249] As an explanation of the above formula, using [Na+] = [0.368] and 50-% formamide, with a GC content of 42%, the average probe size is 200 bases, T m The temperature is 57℃. With each 1% decrease in homology, the T0 of the DNA double helix... m The temperature is reduced by 1-1.5°C. Therefore, using a hybridization temperature of 42°C, targets with greater than approximately 75% sequence identity can be observed. Such sequences will be considered substantially homologous to the nucleic acid sequences of this invention.

[0250] It is well known in the art to gradually increase the stringency of hybridization until only a few positive clones remain. For detection of approximately 80%–90% identical sequences, other suitable conditions include, for example, overnight hybridization at 42°C in 0.25M Na₂HPO₄ (pH 7.2), 6.5% SDS, and 10% dextran sulfate, followed by a final wash at 55°C in 0.1X SSC and 0.1% SDS. For detection of more than approximately 90% identical sequences, suitable conditions include overnight hybridization at 65°C in 0.25M Na₂HPO₄ (pH 7.2), 6.5% SDS, and 10% dextran sulfate, followed by a final wash at 60°C in 0.1X SSC and 0.1% SDS.

[0251] In another embodiment, the hybridization of a nucleic acid molecule with a variant can be determined or identified indirectly, for example, by using nucleic acid amplification reactions, particularly polymerase chain reaction (PCR). PCR requires the use of two primers to specifically amplify the target nucleic acid, and therefore preferably uses two nucleic acid molecules having sequences specific to the QA-3-O-TriS gene of this invention. With RACEPCR, only one such primer may be required (see "PCR protocols; A Guide to Methods and Applications", edited by Innis et al., Academic Press, New York, (1990)).

[0252] Therefore, methods involving obtaining nucleic acids according to the present invention using PCR may include:

[0253] (a) A preparation providing, for example, plant nucleic acids from seeds or other suitable tissues or organs,

[0254] (b) Provide a pair of nucleic acid primers that can be used (i.e., are suitable for) PCR, at least one of which is a primer according to the invention as discussed above.

[0255] (c) Under PCR conditions, the nucleic acid in the preparation is contacted with the primer.

[0256] (d) Perform PCR and determine the presence of amplified PCR products. The presence of amplified PCR products may indicate the identification of a variant.

[0257] In all of the above cases, the clones or fragments identified in the search can be expanded if needed. For example, if they are suspected to be incomplete, the original DNA source (e.g., clonal library, mRNA preparation, etc.) can be revisited to isolate the missing portion, for example, by using sequences, probes, or primers based on the obtained portion to identify other clones containing overlapping sequences.

[0258] Purified proteins (peptides, enzymes) or fragments thereof, mutants, derivatives or variants (e.g., generated by recombination through expression of their encoded nucleic acids) constitute an aspect of the present invention.

[0259] These purified peptides can be used to generate antibodies using standard techniques in the art. As discussed further below, antibodies and peptides containing antigen-binding fragments of antibodies can be used to identify homologs from other species.

[0260] Methods for generating antibodies include immunizing mammals (e.g., humans, mice, rats, rabbits, horses, goats, sheep, or monkeys) with the said protein or fragments thereof. Antibodies can be obtained from the immunized animal using any of a variety of techniques known in the art, and antibodies can be screened, preferably using antibodies that bind to the target antigen. For example, Western blotting or immunoprecipitation can be used (Armitage et al., 1992, Nature 357:80-82). Antibodies can be polyclonal or monoclonal.

[0261] As an alternative or supplement to immunizing mammals, antibodies with appropriate binding specificity can be obtained from recombinant libraries of expressed immunoglobulin variable domains, for example using λ phages or filamentous phages that display functional immunoglobulin binding domains on their surfaces; see, for example, WO 92 / 01047.

[0262] Antibodies generated against polypeptides or peptides can be used to identify and / or isolate homologous polypeptides, which can then be used to encode genes.

[0263] Antibodies can be modified in a variety of ways. In fact, the term "antibody" should be interpreted to encompass any specific binding substance with a binding domain possessing the desired specificity. Therefore, the term covers antibody fragments, derivatives, functional equivalents, and antibody homologs, including any polypeptide containing an immunoglobulin-binding domain, whether natural or synthetic.

[0264] ***

[0265] This document cites numerous patents and publications to provide a more comprehensive description and disclosure of the invention and the level of expertise in the field to which it pertains. Each of these references is incorporated herein by reference in its entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

[0266] Throughout this specification (including the claims thereafter), unless the context otherwise requires, the word “comprise” and variations such as “comprises” or “comprising” shall be understood to mean including one or more of the mentioned whole or steps, but not excluding any other whole or steps or groups of whole or steps.

[0267] It must be noted that, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a drug carrier” includes a mixture of two or more such carriers, etc.

[0268] Ranges are frequently expressed herein as "about" a particular value and / or "about" another particular value. In expressing such ranges, another embodiment includes from a particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent "about," it should be understood that the particular value constitutes another embodiment.

[0269] Any subheadings are included herein for convenience only and should not be construed as limiting this publication in any way.

[0270] The invention will now be further described with reference to the following non-limiting drawings and embodiments. In view of these, those skilled in the art will conceive of other embodiments of the invention.

[0271] abbreviation

[0272] Compound 1–3β-{[α-L-pyranorhamnosyl-(1->3)-[β-D-pyranogalactosyl-(1->2)]-β-D-pyranoglucoside]oxy}-saponin acid

[0273] Compound 2–3β-{[β-D-xylanopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponin acid

[0274] Galp–D-galactopyranose

[0275] GlcpA–D-glucopyranoside (the additional number indicates a specific carbon atom, i.e., GlcA-1)

[0276] GmSGT2 / GmUGT73P2 – Soybean (Glycine max / soybean) soybean saponins β-D-galactosyltransferase GT – glycosyltransferase

[0277] QA – Saponin Acid

[0278] QA-GlcpA–3β-{[β-D-glucopyranoside]oxy}-saponin acid

[0279] QA-GlcpA-Galp–3β-{[β-D-galactopyranosyl-(1->2)-β-D-glucopyranoside]oxy}-Saponin Acid QA-GlcpA-[Galp]-Rhap–3β-{[α-L-rhamnosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-Saponin Acid

[0280] QA-GlcpA-[Galp]-Xylp–3β-{[β-D-xylanopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponin acid

[0281] QA-3-O-TriS– is a QA glycosylated with a branched trisaccharide at the 3-O position, which is either QA-GlcpA-[Galp]-Rhap or QA-GlcpA-[Galp]-Xylp.

[0282] OS-2,3-Oxysqualene

[0283] ***

[0284] QA-GlcAT–QA 3-O-glucuronyltransferase

[0285] QA-GalT–QA-GlcpA galactosyltransferase

[0286] QA-RhaT / XylT–QA-GlcpA-Galp rhamnosyl and / or xylosyltransferase

[0287] QsbAS – Saponin Synthase

[0288] Qs-3-O-GalT–Saponin QA-GlcpAβ-1,2-D-galactosyltransferase

[0289] QsCSL1 – Saponin cellulose synthase-like enzyme (soapberry acid 3-O-glucuronyl transferase)

[0290] QsCslG2–Saponin cellulose synthase-like enzyme (Saponin acid 3-O-glucuronyl transferase)

[0291] Qs-3-O-RhaT / XylT–Saponin QA-GlcpA-Galp Dual β-1,3-D-Xylosyltransferase / α-1,3-L-Rhamnosyltransferase

[0292] Qs_0283850-Soap Tree QA-GlcpA-Galpα-1,3-L-Rhamnosyltransferase

[0293] DN20529_c0_g2_i8-Saponin QA-GlcpA-Galpα-1,3-L-Rhamnosyltransferase

[0294] Qs_0283870- Soap tree QA-GlcpA-Galpβ-1,3-D-xylosyltransferase

[0295] QsCYP716-C-28 – Soapberry saponin C-28 oxidase

[0296] QsCYP716-C-16α–Saponin acid C-16α oxidase

[0297] QsCYP714-C-23 – Soapberry saponin C-23 oxidase

[0298] Rhap–L-rhamnopyranose

[0299] tHMGR – truncated 3-hydroxy,3-methylbutyryl-CoA reductase from Avena strigosa (diploid oat)

[0300] Family 1 UGT – Family 1 UDP-dependent glycosyltransferases

[0301] Xylp–D-xylanose

[0302] Qs_2073886_D6 – Synonymous with Qs-3-O-GalT

[0303] Qs_2015879_D7 – Synonymous with Qs-3-O-RhaT / XylT Attached Figure Description

[0304] Figure 1: QS-21. Highlights key structural features, including the saponin triterpenoid glycoside agligand, the branched trisaccharide at C-3, the linear tetrasaccharide at C-28, and the arabinoglycosylated 18-carbon acyl chain of β-D-fucose attached to C-28.

[0305] Figure 2: Production of saponin acid from 2,3-oxidized squalene via β-caryophyllin. Important β-caryophyllin carbons mentioned herein are marked in red. The pathway from β-caryophyllin requires oxidation at three positions (C-28, C-23, and C-16α). For simplicity, these oxidation steps are shown linearly, but they can occur in any order.

[0306] Figure 3: Production of saponin trisaccharide derivative (QA-3-O-TriS) from saponin acid. The pathway (shown in linear order for simplicity) requires a three-step glycosylation starting at C-3 of saponin acid with D-glucuronic acid (GlcpA). GlcpA is further glycosylated by β-1,2-D-galactose (Galp) and β-1,3-D-xylose (Xylp) in one or another sequence.

[0307] Figure 4: Discovery of candidate QS-21UDP-dependent glycosyltransferases (UGTs). Phylogenetic tree of soapberry UGT candidates (red) and characterized UGTs (black) from other plant species (listed in Table 3). Triterpenoid UGTs with functional characteristics are shown in blue. Soapberry UGTs whose genes are predicted to be within the biosynthetic gene cluster (BCG) are indicated by an asterisk. The UGT phylogenetic group (group AP) was labeled as follows: Ross, J., Li, Y., Lim, E., and Bowles, DJ (2001). "Higher plant glycosyltransferases". Genome Biol., 2: REVIEWS 3004; and Caputi, L., Malnoy, M., Goremykin, V., Nikiforova, S., and Martens, S. (2012). "A genome-wide phylogenetic reconstruction of family 1UDP-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 a neighbor-joining method with 1000 bootstrap copies (showing the percentage of support at branch points). The scale bar shows 0.1 substitutions at each site at the amino acid level. The soapberry contiguous group in the 1KP database consists of a 4-letter code (OQHZ) followed by a seven-digit number; this seven-digit code is included in the name of the candidate UGT gene.

[0308] Figure 5: Conversion of saponin by QsCSL1. Accumulation of saponin was detected in leaves expressing QsbAS and C-28 / C-23 / C-16α oxidases. Addition of saponin cellulose synthase-like enzyme (QsCSL1) resulted in lower levels of saponin, and a new peak in saponin mass accumulation upon addition of glucuronide residues [m / z = 661, retention time = 13.9 min]. IS = internal standard (digitoxin).

[0309] Figure 6: GmUGT73P2 (accession number: BAI99584) from soybean catalyzes the addition of D-galactose to soybean saponin B monoglucuronic acid with β-1,2-linked structure to form soybean saponin III (Shibuya et al., 2010).

[0310] Figure 7: Transformation of QA-GlcpA via Qs_2073886_D6 (Qs-3-O-GalT). Co-expression of Qs_2073886_D6 with the genes required for the production of putative QA-GlcpA (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCSL1) resulted in a larger polar peak [m / z = 823, retention time = 12.6 min] of QA-GlcpA with added hexose. The mean mass spectrum of this peak is shown. Co-expression of the genes required for the production of 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).

[0311] Figure 8: Co-expression of Qs_2015879_D7 with the genes required to generate the putative QA-GlcpA-Galp (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCSL1 / Qs-3-O-GalT) resulted in two co-eluting peaks with retention times similar to the putative QA-GlcpA-Galp peak. The more polar peak has the expected mass of QA-GlcpA-Galp with added deoxyhexose [m / z = 869, retention time = 12.5 min]; the less polar peak has the expected mass of QA-GlcpA-Galp with added pentose [m / z = 855, retention time = 12.75 min]. The mean mass spectra and predicted structures for each peak are shown. Co-expression of the combination without the Qs-3-O-GalT gene did not result in any new peaks, indicating that Qs_2015879_D7 depends on Qs-3-O-GalT activity. IS = Internal Standard (Digitatoxin).

[0312] Figure 9: Biosynthesis of the proposed QA-GlcpA-[Galp]-Rhap and QA-GlcpA-[Galp]-Xylp from saponin using the enzymes characterized in this paper.

[0313] Figure 10: Structures of isolated compounds 1 and 2.

[0314] Figure 11: Complete reports for compounds 1 and 2 13 C NMR allocation and key HMBC (H→C)

[0315] Figure 12: Schematic diagram of the separation of trisaccharides from tobacco leaf material.

[0316] Figure 13: Semi-preparative HPLC chromatograms used for the purification of compounds 1 and 2.

[0317] Figure 14: A heatmap showing the expression profile of the previously characterized QS-21 biosynthetic genes.

[0318] Figure 15: Phylogenetic tree of saponin cellulose synthase superfamily proteins (bold) and cellulose synthase superfamily proteins from rice (Oryza sativa) and Arabidopsis thaliana. The tree was constructed using a neighbor-joining method with 1000 bootstrap copies (showing the percentage of support at branch points). Saponin genes in the genome database consist of a code (QUISA32244_EIv1_) followed by seven digits; this seven-digit code is included in the name of the saponin protein; saponin proteins belonging to the same subfamily (CslG) as CSL1 have been renamed CslG2-6.

[0319] Figure 16: A heatmap showing the expression profiles of the previously characterized QS-21 biosynthetic gene and CslG2-CslG6.

[0320] Figure 17: Co-expression of CslG2 with genes used to produce saponin (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23) resulted in a decrease in the saponin peak [m / z = 485, retention time = 19.6 min], and a larger polar peak with the expected quality of saponin appeared with the addition of glucuronide residues, and had the same retention time as the CSL1 product [m / z = 661, retention time = 14.0 min]. IS = internal standard (digitoxin).

[0321] Figure 18: Qs-3-O-RhaT / XylT is a chimera between two adjacent genes Qs_0283860 and Qs_0283870 in the soapberry genome. The soapberry gene in the genome database consists of a code (QUISA32244_EIv1) followed by seven digits; this seven-digit code is included in the name of the soapberry gene.

[0322] Figure 19: Transformation of QA-GlcpA-Galp using Qs_0283850, DN20529_c0_g2_i8 and Qs_0283870. Co-expression of the bifunctional Qs-3-O-RhaT / XylT with the gene (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCSL1 / Qs-3-O-GalT) for the production of QA-GlcpA-Galp resulted in the conversion of QA-GlcpA-Galp (retention time = 12.6 min, MW = 824) into 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 gene required for the production of QA-GlcpA-Galp resulted in a decrease in the QA-GlcpA-Galp peak at 12.6 min followed by the appearance of a single new peak, which had the same retention time (12.5 min) and molecular weight (MW = 970) as QA-GlcpA-[Galp]-Rhap. Co-expression of Qs_0283870 with the gene required for the production of QA-GlcpA-Galp resulted in a decrease in the QA-GlcpA-Galp peak at 12.6 min followed by the accumulation of a single new peak, which had the same retention time (12.75 min) and molecular weight (MW = 956) as QA-GlcpA-[Galp]-Xylp.

[0323] Example

[0324] Example 1 - Identification and Cloning of Glycosyltransferases from Soap Trees

[0325] To increase the publicly available transcriptome, we generated genome sequence data (PacBio sequencing performed by Earlham Institute, Norwich, Norfolk). The genome sequences were annotated using publicly available data (including transcriptome data from '1KP' leaves of soapberry [4]) and proteins from related plant species in the Phytozome.

[0326] Based on these data, we screened a series of sequences annotated as presumed family 1UDP-dependent glycosyltransferases (UGTs), an important class of enzymes known to be involved in the biosynthesis of many plant natural products, including triterpenes [5, 6]. We refined the initial list (containing approximately 200 sequences) to the following sequences, which also appeared in the 1KP database, from which the original QA biosynthetic enzymes were identified. The soapberry contig is a 4-letter code (OQHZ) followed by a seven-digit number. Where possible, all the following candidate genes include this seven-digit code. To further refine this list, we performed a phylogenetic analysis using a series of characterized GTs from other plant species (Table 3). This led us to prioritize enzymes belonging to the same phylogenetic group as the currently characterized triterpenoid UGTs (groups A, D, and L) from other plant species and UGTs with associated sugar donor specificity (group B).

[0327] Finally, in recent years, it has been proposed that many chemically distinct plant natural products are synthesized by enzymes encoded by physically colocalized genes. These so-called 'biosynthetic gene clusters' (BCGs) can facilitate the identification of other candidate genes. Therefore, we deployed the 'PlantiSMASH' genome mining tool [7] to predict possible BCGs in the soapberry genome. This combination of methods produced 30 candidate soapberry UGTs (Figure 4) plus a final list of other non-UGT candidate genes.

[0328] As mentioned above, the gene for saponin biosynthesis appears to be expressed in leaf tissue and was previously amplified from leaf cDNA by PCR. Therefore, the same method was used to amplify the GT candidate. A series of oligonucleotide primers were designed with incorporation at the 5' attB site upstream of the target sequence to allow… Cloning. The gene was successfully amplified and cloned into pDONR 207. The clone was sequenced and then transferred into the plant expression vector pEAQ-HT-DEST1

[14] . Finally, the expression construct was transformed into Agrobacterium tumefaciens (LBA4404) for transient expression in Nicotiana benthamiana.

[0329] Thirty-one candidate GTs were screened using transient expression in *Nicotiana benthamiana*. All infiltrations included four *Agrobacterium tumefaciens* strains carrying constructs for QA biosynthesis (QsbAS and C-28 / C-23 / C-16α oxidases) and a strain carrying tHMGR (a key yield-enhancing enzyme for triterpenoid production).

[0330] Example 2 - Identification of saponin 3-O-glucuronyltransferase

[0331] Unexpectedly, after LC-MS analysis of the sample, a candidate, a predicted 'cellulosic synthase-like' (CSL) enzyme (named QsCSL1 in this paper), was found to be active for saponin acid. Co-expression of this enzyme with five Agrobacterium tumefaciens species used to produce QA resulted in a significant loss of the QA peak at 19.2 min, with a new peak appearing at 13.9 min (Figure 5). The change in peak retention indicated a significant increase in polarity, as expected by the addition of sugar. Furthermore, MS analysis of the peak showed a mass number of 662, consistent with the predicted molecular weight of saponin acid glucuronide (Figure 5). As previously described

[19] , we performed large-scale percolation of *Nicotiana benthamiana* to purify a sufficient amount of the compound (68.1 g) and then determined its structure by NMR. This confirmed that the compound was 3β-{[β-D-glucopyranoside]oxy}-saponin acid (QA-GlcpA) (Table 11).

[0332] Example 3 - Identification of QA-GlcpA galactosyltransferase

[0333] After identifying the proposed glucuronyltransferase, the next proposed step is to add β-D-galactose residues.

[0334] The triterpenoid 3-O-glucuronide-β-1,2-galactosyltransferase GmUGT73P2 was previously identified in soybean (soybean / Glycine max) (Shibuya et al., 2010). This enzyme catalyzes the addition of D-galactose to soybean saponin β-monoglucuronide to form soybean saponin III (Figure 6).

[0335] Interestingly, phylogenetic analysis of saponin UGT enzymes revealed a candidate, Qs_2073886_D6, closely associated with GmUGT73P2 (Figure 4). Analysis of the predicted protein sequence of this candidate also indicated that it possesses histidine residue signatures of galactosyl or arabinosyltransferases (Table 4) (Kubo et al., 2004; Han et al., 2014; Louveau et al., 2018). Therefore, Qs_2073886_D6 was preferentially considered as a possible galactosyltransferase.

[0336] Qs_2073886_D6 is co-expressed with the six genes required to produce the putative QA-GlcpA (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCSL1). HPLC-MS analysis indicated that Qs_2073886_D6 appeared to convert the putative QA-GlcpA product into a new, more polar product at 12.6 min (Figure 7, top). MS analysis of this product showed a mass number of 824, consistent with the addition of a hexose (e.g., galactose) (Figure 7, bottom).

[0337] To further confirm the identity of the novel product, we used the soybean (Glycine max) triterpenoid 3-O-glucuronide-β-1,2-galactosyltransferase GmUGT73P2. It was hypothesized that this enzyme might exhibit similar galactosyltransferase activity to the proposed QA-GlcpA product. Therefore, permeation was also performed by co-expressing six enzymes necessary for the synthesis of QA-GlcpA and GmUGT73P2. LC-MS analysis of the permeated leaf extracts showed that a peak was indeed observed in the sample expressing GmUGT73P2, which had a matching retention time and mass spectrum with the product observed at 12.6 min in the sample expressing saponin galactosyltransferase (Figure 7, top). This provides substantial evidence that the saponin enzyme possesses triterpenoid 3-O-glucuronide-β-1,2-galactosyltransferase activity to form the saponin disaccharide QA-GlcpA-Galp. Therefore, the enzyme is named Qs-3-O-GalT in this paper. Qs-3-O-GalT and GmUGT73P2 share 68% sequence identity at the nucleotide level and 57% sequence identity at the protein level.

[0338] We also performed large-scale tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCSL1 / Qs-3-O-GalT percolation in *Nicotiana benthamiana*, as previously described

[19] , to purify the compound (32.1 g), and subsequently determined its structure by NMR. This confirmed that the compound is 3β-{[β-D-galactopyranosyl-(1->2)-β-D-glucopyranoside]oxy}-saponin (QA-GlcpA-Galp) (Table 13).

[0339] Example 4 - Identification of QA-GlcpA-Galp dual rhamnosyl / xylosyltransferase

[0340] Next, we repeated the method of screening remaining GT candidates for the QA-GlcpA-Galp product. As previously described, GT candidates were screened by co-expression with the seven genes required to produce QA-GlcpA-Galp (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCSL1 / Qs-3-O-GalT). Using this strategy, we identified the UGT enzyme that caused the depletion of the QA-GlcpA-Galp product. However, instead of observing a single new product, we observed the appearance of two new products with retention times very close to those of the previous QA-GlcpA-Galp (Figure 8). The mass spectra of each product were unique; the first (retention time 12.50 min, Figure 8, bottom left) indicated a mass number of 970, and the second (retention time 12.8 min, Figure 8, bottom right) indicated a mass number of 956. Compared to the QA-GlcpA-Galp product (MW=824), peaks 1 and 2 will be consistent with the addition of deoxyhexose and pentose, respectively.

[0341] It is known that soap trees produce more than 100 different saponins

[16] . Among these saponins, the 3-O-GlcpA-β-1,2-D-Galp disaccharide of QA is highly conserved

[17] , while the terminal sugar attached at the C3 position of GlcpA in the branched trisaccharide of QA is altered. The alteration is the addition of α-L-rhamnose (Rhap-a deoxyhexose) or β-D-xylose (Xylp-a pentose)

[17] , the latter of which was observed in QS-21 (Fig. 1). Thus, the new compounds observed in *Benjamina baccata* are consistent with the addition of Rhap or Xylp at the 3-O position of GlcA in the QA-Glcp-Galp disaccharide. These compounds were named QA-GlcpA-[Galp]-Rhap(3β-{[α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponic acid) and QA-GlcpA-[Galp]-Xylp(3β-{[β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranoside]oxy}-saponic acid). The isolation and structural verification of these compounds are described below. The glycotransferases are referred to as Qs-3-O-RhaT / XylT, and Figure 9 shows a schematic diagram of the biosynthesis of these two trisaccharides, QA-GlcpA-[Galp]-Rhap and QA-GlcpA-[Galp]-Xylp.

[0342] Previously, chemical analysis of soapberry trees has shown the existence of different 'chemotypes' that differ in their ability to produce saponins containing Rhap or Xylp attached to GlcpA-3-O (see WO 2018 / 057031). One explanation for these observations is the presence of two distinct alleles of terminal glycotransferases (with different sugar specificities), as previously demonstrated in soybeans

[18] . Nevertheless, this disclosure provides an enzyme capable of catalyzing the addition of two different sugars at the same position.

[0343] Example 5 - Purification and NMR Verification of Trisaccharides Produced in Nicotiana benthamiana

[0344] To verify the structures of compounds 1 and 2 (Fig. 10), we performed large-scale infiltration of *Nicotiana benthamiana* plants as previously described

[19] . Plants were infiltrated with *Agrobacterium tumefaciens* strains carrying eight pEAQ-HT-DEST1 constructs (tHMGR, QsbAS, CYP716-C-28, CYP716-C-16α, CYP714-C-23, QsCSL1, Qs-3-O-GalT, and Qs-3-O-RhaT / XylT) for the production of these two trisaccharides. After harvesting and subsequent isolation, we successfully obtained 1.8 and 0.9 mg of purified compounds 1 and 2, respectively. Subsequent steps were then performed... 1 H and 13 C10 NMR analysis confirmed the identity of compound 1 (3β-{[α-L-pyranoraminosyl-(1->3)-[β-D-pyranogalactosyl-(1->2)]-β-D-pyranoglucopyranoside]oxy}-saponic acid) and compound 2 (3β-{[β-D-pyranoxyl-(1->3)-[β-D-pyranogalactosyl-(1->2)]-β-D-pyranoglucopyranoside]oxy}-saponic acid) (Figure 10). NMR partitions are given in Table 1 and Figure 11.

[0345] Example 6 – Using the QA-3-O-TriS gene, optionally combined with the QA gene, to produce plants with stable transformation.

[0346] Triterpenes have previously been produced using engineered transgenic plant lines (e.g., Arabidopsis thaliana, wheat). A series of Golden Gate

[23] vectors have been reported that allow the construction of multi-gene vectors and allow the integration of the entire pathway into a single locus. These can be similarly applied to the present invention based on the disclosure herein.

[0347] Therefore, the QA-3-O-TriS gene described herein, optionally combined with the QA gene in the previously submitted unpublished PCT / EP2018 / 086430 (later published as WO 2019 / 122259), can be used to produce stable transgenic plants in combination with known transgenic technologies according to this disclosure.

[0348] Example 7 – Saponin 3- O Identification of glucuronyltransferase CSLG2 (QsCSLG2)

[0349] As described in the preceding embodiments, the '1KP' soapberry leaf transcriptome was used to identify genes involved in the biosynthesis of saponin (QsbAS, QsCYP716-C-16α, QsCYP714-C-23, and QsCYP716-C-28) and the trisaccharides (QsCSL1, Qs-3-O-GalT, and Qs-3-O-RhaT / XylT) at the C-3 position of QS-21.

[0350] Genes involved in the biosynthesis of triterpenoid glycosides are often co-expressed

[25] . To investigate the expression patterns of the characterized QS-21 biosynthetic gene in multiple tissues, RNA-seq data were generated for six soapberry tissues (primordia, extended leaves, mature leaves, old leaves, green stems, and roots). The gene expression profiles of QsbAS, QsCYP716-C-16α, QsCYP714-C-23, QsCYP716-C-28, and Qs-3-O-GalT showed a pattern of low expression in old leaves and high expression in primordia, with some variation in expression levels in roots, extended leaves, green stems, and mature leaves (Fig. 14). In contrast, the expression profile of QsCSL1 showed the highest expression level in old leaves (Fig. 14). Qs-3-O-RhaT / XylT was not included in this analysis (see Example 8 below).

[0351] Because the expression profile of QsCSL1 does not follow the general pattern of other characterized QS-21 genes, it was investigated whether there might be QsCSL1-related genes that do indeed have a QS-21 gene expression pattern and therefore may be involved in QS-21 biosynthesis. QsCSL1 was used in a BLASTp search to identify cellulose synthase-like genes in the soapberry annotated genome. This identified 39 additional cellulose synthase superfamily genes, five of which (named CslG2 to CslG6) belong to the same subfamily as QsCSL1 (Figure 15).

[0352] Expression profile analysis of these genes showed that CslG3-CslG6 were most highly expressed in older leaves or roots (Figure 16). Interestingly, one gene, CslG2, shared the same expression profile as other QS-21 biosynthetic genes, exhibiting relatively high expression in primordia and relatively low expression in older leaves (Figure 16). This gene shared 78% DNA sequence identity and 70% protein sequence identity with QsCSL1.

[0353] To investigate potential saponin glucuronyl transferase activity, CslG2 was amplified from leaf cDNA, cloned into the plant expression vector pEAQ-HT-DEST1, and transformed into Agrobacterium tumefaciens for transient expression in Nicotiana benthamiana. QsbAS, QsCYP716-C-16α, QsCYP714-C-23, QsCYP716-C-28, and CslG2 were transiently co-expressed in Nicotiana benthamiana. This indicated that CslG2 had the same activity as CSL1: a reduced saponin peak and a larger polar peak of saponin mass upon the addition of glucuronyl ester residues (Fig. 17). As previously described

[19] , we performed large-scale percolation of Nicotiana benthamiana to purify 2.1 mg of the target molecule. This was confirmed by NMR to be 3β-{[β-D-glucuronyl pyranoside]oxy}-saponin (QA-GlcpA) (Table 12).

[0354] Example 8 – QA-Glc p A-Gal p Identification of xylosyltransferase and rhamnosyltransferase

[0355] As illustrated in Example 4, the DNA sequence of the diglycosyltransferase Qs-3-O-RhaT / XylT was not identified in the soapberry genome dataset. Instead, the gene appears to be a chimera between two adjacent genes, Qs_0283860 (pseudogene) and Qs_0283870 (Fig. 18). The portion of the Qs_0283860 pseudogene incorporated into the Qs-3-O-RhaT / XylT sequence shows high sequence similarity to another adjacent gene, Qs_0283850 (Fig. 18, Table 9).

[0356] Theoretically, it is possible that alleles of these genes exist that are not expressed in the soapberry genome dataset, or that this region has been misresolved. As an alternative database, de novo transcriptome components were generated from soapberry primordia RNA-seq reads

[26] . Two full-length transcripts were identified using a BLASTn search with three genomic genes and Qs-3-O-RhaT / XylT as the query sequence: DN20529_c0_g2_i6, which is sequence identical to Qs_0283870 and confirmed to be the sequence of that gene; and DN20529_c0_g2_i8, which has 99% DNA sequence identity with the pseudogene Qs_0283860 and 98% DNA sequence identity with Qs_0283850 (Table 9).

[0357] To investigate the presence and function of these genes, we attempted to amplify sequences from soapberry leaf cDNA. Qs_0283850 and Qs_0283870 were successfully amplified. Primers designed to amplify the pseudogene Qs_0283860 amplified a full-length sequence with 100% sequence identity to the coding region of the gene predicted by the de novo transcriptome DN20529_c0_g2_i8. This amplified sequence was subsequently named DN20529_c0_g2_i8. These three amplified genes (Qs_0283850, Qs_0283870, and DN20529_c0_g2_i8) were cloned into the plant expression vector pEAQ-HT-DEST1 and transformed into *Agrobacterium tumefaciens* for transient expression in *Nicotiana benthamiana*.

[0358] As described above, co-expression of Qs-3-O-RhaT / XylT with seven genes (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCSL1 / Qs-3-O-GalT) that can produce QA-GlcpA-Galp results in the trisaccharide QA-GlcpA-[Galp]-Rhap (retention time = 12.5 min, MW = 970) and QA-GlcpA-[Galp]-Xylp (retention time = 12.75 min, MW = 956), which have retention times very close to those of the previous QA-GlcpA-Gal (retention time = 12.6 min, MW = 824) (Figure 19).

[0359] Similarly, co-expression of Qs_0283850, Qs_0283870, or DN20529_c0_g2_i8 with the gene required for the production of QA-GlcpA-Galp indicates that all three enzymes can convert QA-GlcpA-Galp, but each enzyme results in the production of a novel product (Figure 19). Qs_0283850 and DN20529_c0_g2_i8 exhibit similar activity, reducing the QA-GlcpA-Galp peak and accumulating a larger polar peak with the same retention time (12.5 min) and molecular weight (MW = 970) as QA-GlcpA-[Galp]-Rhap (Figure 19). This indicates that Qs_0283850 and DN20529_c0_g2_i8 possess rhamnosyltransferase activity and can produce QA-GlcpA-[Galp]-Rhap as a single product without producing QA-GlcpA-[Galp]-Xylp. Large-scale infiltration was performed on Nicotiana benthamiana

[19] to transiently express QsbAS, QsCYP716-C-16α, QsCYP714-C-23, QsCYP716-C-28, CslG2, Qs-3-O-GalT and Qs_0283850. Purification of the product (43.3 mg) and structural analysis by NMR confirmed that its structure is 3β-{[α-L-pyranoraminosyl-(1->3)-[β-D-pyranogalactosyl-(1->2)]-β-D-pyranoglucopyranoside]oxy}-saponin acid (QA-GlcpA-[Galp]-Rhap) (Table 14).

[0360] Co-expression of Qs_0283870 with the gene required to produce QA-GlcpA-Galp also reduced the QA-GlcpA-Galp peak, but it accumulated a smaller polar compound with the same retention time (12.75 min) and molecular weight (MW = 956) as QA-GlcpA-[Galp]-Xylp (Fig. 6). Large-scale infiltration was performed on Nicotiana benthamiana plants

[19] to co-express tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / QsCskG2 / Qs-3-O-GalT / Qs_0283870. Purification of the obtained compound (21.6 mg) and structural analysis by NMR confirmed that its structure is 3β-{[β-D-pyranoxyl-(1->3)-[β-D-pyranogalactosyl-(1->2)]-β-D-pyranoglucoside]oxy}-saponin acid (QA-GlcpA-[Galp]-Xylp) (Table 15).

[0361] This indicates that Qs_0283870 is primarily a xylosyltransferase and can produce QA-GlcpA-[Galp]-Xylp without producing large amounts of QA-GlcpA-[Galp]-Rhap.

[0362] Materials and Methods

[0363] Phylogenetic analysis of UGT candidates

[0364] Amino acid sequences were inferred from the predicted full-length coding sequence of the soapberry UGT. Representative amino acid sequences of the glycosyltransferase family 1UGT from other plant species (Table 3) were obtained from the NCBI database and included in the phylogenetic analysis. Protein sequences were aligned using MAFFT (https: / / mafft.cbrc.jp / alignment / software / ). The rootless tree was constructed in MEGA7 using the neighbor-joining method with 1000 bootstrap replicas [20, 21].

[0365] Primers and Cloning

[0366] Genes encoding the enzymes described herein (QsCSL1, Qs-3-O-GalT, Qs-3-O-RhaT / XylT, QsCslG2, Qs_0283850, DN20529_c0_g2_i8, and Qs_0283870) were amplified from cDNA derived from soapberry leaf tissue by PCR. PCR was performed using iProof polymerase with thermal cycling as recommended by the manufacturer, using primers detailed in Tables 2 and 10. The resulting PCR products were purified (Qiagen PCR Clean Kit) and cloned individually into the pDONR207 vector using BP clonase according to the manufacturer's instructions. The BP reaction was transformed into *E. coli*, and the resulting transformants were cultured and plasmids isolated using miniprep (Qiagen). The isolated plasmids were sequenced (Eurofins) to verify the presence of the correct genes. Next, each of the three genes was further subcloned into the pEAQ-HT-DEST1 expression vector using LR clonase. The resulting vector was transformed into Agrobacterium tumefaciens LBA4404 by rapid freezing in liquid N2.

[0367] Agrobacterium tumefaciens penetration from tobacco leaves

[0368] As previously described

[19] , Agrobacterium infiltration was performed using a needle-free injector. As described above, all genes were expressed in Agrobacterium tumefaciens LBA4404 from the pEAQ-HT-DEST1 binary expression vector

[14] . Culture of the bacteria and plants was as described in

[19] .

[0369] Preparation of Nicotiana benthamiana leaf extract for LC-MS analysis

[0370] Leaves were harvested after 5 days of Agrobacterium infiltration and then freeze-dried. The freeze-dried leaf material (10 mg / sample) was milled at 1000 rpm for 1 min (Geno / Grinder 2010, Spex SamplePrep). Extraction was performed in 550 μL of 80% methanol with 20 μg / mL digoxin (internal standard; Sigma) at 40 °C for 20 min with shaking at 1400 rpm (Comfort thermostat, Eppendorf). The sample was partitioned twice with 400 μL of hexane. The aqueous phase was dried under vacuum at 40 °C (EZ-2 series evaporator, Genevac). The dried material was resuspended in 75 μL of 100% methanol and filtered at 12,500 g for 30 seconds (0.2 μm, Spin-X, Costa). The filtered sample was transferred to glass vials and analyzed as detailed below.

[0371] LC-MS analysis of tobacco leaf extract

[0372] Analysis was performed using a Prominence HPLC system equipped with a single quadrupole mass spectrometer LCMS-2020 (Shimadzu) and a Corona Veo RS charged electrosol detector (CAD) (Dionex). Detection: MS (dual ESI / APCI ionization, DL temperature 250℃, spray gas flow rate 15 L / min, heating block temperature 400℃, spray positive voltage 4.5 kV, negative voltage 3.5 kV); CAD: data acquisition rate 10 Hz, filtration constant 3.6 s, 925 evaporator temperature 35℃, ion trap voltage 20.5 V. Method: Solvent A: [H₂O + 0.1% formic acid], Solvent B: [acetonitrile (CH₃CN) + 0.1% formic acid]. Injection volume: 10 μL. Gradients: 15% [B] from 0 to 1.5 min, 15% to 60% [B] from 1.5 to 26 min, 60% to 100% [B] from 26 to 26.5 min, 100% [B] from 26.5 to 28.5 min, 100% to 15% [B] from 28.5 to 29 min, 35% [B] from 29 to 30 min. A flow rate of 0.3 mL / min was used with a Kinetex 2.6 μm XB-C18 column. The 50x2.1mm (Phenomenex) sample was analyzed using LabSolutions software (Shimadzu).

[0373] Large-scale vacuum infiltration of Benedictine tobacco

[0374] As previously described [19, 22], a total of 198 plant species were vacuum-permeated using Agrobacterium rhizogenes strains carrying the pEAQ-HT-DEST1 construct targeting tHMGR, QsbAS, CYP716-C-28, CYP716-C-16α, CYP714-C-23, QsCSL1, Qs-3-O-GalT, and Qs-3-O-RhaT / XylT. The plants were harvested after 4 days and freeze-dried to obtain a total of 175.25 g of dried leaf material.

[0375] Purification of compounds from large-scale infiltration of tobacco Benzodiaceae.

[0376] General Program

[0377] The organic solvents used for extraction and rapid chromatography are reagent-grade and can be used directly without further distillation. The HPLC mobile phase is prepared using HPLC-grade solvents. Kinetex-XB-C is used. 18 LC-MS spectral data were recorded on a SHIMADZU-2020 (single quadrupole) instrument (50×10mm id; 2.6μm; USA) (JIC, UK). 1D and 2D NMR spectra were recorded on a Bruker Avance 600MHz spectrometer equipped with a BBFO Plus smart probe and a triple resonance TCI cryoprobe (JIC, UK), respectively. Chemical shifts were correlated with residual solvent signals (MeOH-d4:δ). H 3.31; δ C 49.15). When using Luna C 18 Preparative HPLC experiments were performed on an Ultimate 3000 column (250 × 10 mm id; 5 μm; USA). Rapid column chromatography (FCC) was performed using an Isolera One (Biotage) SNAP Ultra 50 g column. Analytical TLC experiments were performed on silica gel pre-coated aluminum plates (F254, 20 × 20 cm, Merck KGaA, Germany). The TLC plates were observed under UV light (254 nm) and then stained with p-anisaldehyde (2% v / v p-anisaldehyde, 2% v / v concentrated H₂SO₄).

[0378] Extraction and separation

[0379] Dry tobacco powder was mixed with quartz sand (0.3–0.9 mm). This mixture was placed on top of a 3 cm deep layer of quartz sand (0.3–0.9 mm) in a 120 mL extraction chamber. Extraction was performed using a rapid extractor E-914 (Büchi) with three cycles at 100 °C and 130 bar. The hold time for cycle one was zero, and the hold times for cycles two and three were 5 min. The run ended with a 1 min solvent rinse and a 12 min N2 rinse. The dried leaves were initially extracted with hexane for defatting, followed by a thorough extraction with methanol. The organic layers were combined and evaporated under reduced pressure. The crude methanol extract was dissolved in a minimal amount of methanol and diluted with an equal volume of water, then successfully partitioned against hexane, dichloromethane, ethyl acetate, and n-butanol using a separatory funnel. The butanol layer was collected again and dried over anhydrous NaSO4, evaporated under reduced pressure, and subjected to normal-phase silica gel rapid chromatography (35-70 μm) using a long gradient of DCM / MeOH [100 / 0-0 / 100] over 30 min. The column was further washed with ethyl acetate / acetone / water / formic acid (5 / 3 / 0.5 / 0.5). All fractions were monitored by TLC using different eluent systems and grouped together according to their polarity. Analysis was also based on LC-MS. 1 ¹H NMR, introducing promising fractions using an elution system of water / acetonitrile containing 0.1% formic acid, via reversed-phase (preparative / semi-preparative C) 18 Further preparative chromatographic purification was performed using HPLC to ultimately provide pure saponins. Detailed separation protocols for the isolated compounds used to purify compounds 1 and 2 are given (see Examples 4 and 5) and the quantities of said compounds are shown (Figure 12). The trisaccharide compounds described in Example 8 were purified using the same method. For the monosaccharide and disaccharide compounds described in Examples 2, 3, and 7, extraction and separation were performed as above, with the following changes: liquid-liquid partitioning was performed against ethyl acetate, the organic layer was dried over anhydrous MgSO4, and the saponin fraction was subsequently purified by reversed-phase C18 HPLC.

[0380] NMR analysis

[0381] Unless otherwise stated, in Fourier transform mode, in relation to 1 600MHz H NMR and targeting 13NMR spectra were recorded in deuterated methanol at a nominal frequency of 150 MHz for C NMR. Chemical studies of the n-butanol fraction of tobacco leaves (Examples 4 and 5) provided the separation of two previously reported triterpenoid saponins, namely 3β-{[α-L-pyranoraminosyl-(1->3)-[β-D-pyranogalactosyl-(1->2)]-β-D-pyranoglucopyranoside]oxy}-saponic acid (1) and 3β-{[β-D-pyranoxylosyl-(1->3)-[β-D-pyranogalactosyl-(1->2)]-β-D-pyranoglucopyranoside]oxy}-saponic acid (2) (Fig. 10). Their structures were resolved based on a combination of spectroscopic tools, including a broad suite of 2D-NMR, mass spectrometry and those reported in the literature

[23] (Table 1 and Fig. 11).

[0382] RNA sequence alignment and heatmap

[0383] RNA-seq data (Illumina sequencing reads) were compared with the soapberry genome using the STAR package (version 2.5)

[27] , and the featureCounts program was used ( http: / / subread.sourceforge.net Quantization was performed using version 1.6.0. The heatmap was plotted in R using heatmap.2. https: / / CRAN.R-project.org / package= gplots ).

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[0412] Tables and sequences

[0413]

[0414]

[0415]

[0416] Table 1: 1H, 1 13C NMR spectral data of Compounds 1 and 2 in MeOH-d4 (600, 150 MHz) 13

[0417] ***

[0418]

[0419] Table 2: Primers used for cloning three glycosyltransferases required for the biosynthesis of the C-3 trisaccharide in saponin. Gene-specific sequences are shown in black. The attB site required for cloning is shown in red.

[0420] ***

[0421]

[0422] Table 4: Alignment of UGT protein sequences in the motif region of plant secondary product glycosyltransferase (PSPG) of family 1UGT 44 amino acids. Qs_2073886_D6 (“Qs-3-O-GalT”) has conserved histidine residues in UGTs that transfer β-D-galactose or α-L-arabinose. Figure adapted from Louveau, Orme

[15] . Accession numbers: AsUGT99A6 (AZQ26916), MtUGT73K1 (AAW56091), AtUGT78D2 (NP_197207), GmSSAT (XP_003532274), AtUGT78D3 (NP_197205), AcGaT (BAD06514), GmUGT73P2 (BAI99584).

[0423]

[0424] Table 5: Glycosyltransferases identified in this paper (“Qs QA-3-O-TriS sequence”):

[0425]

[0426] Table 6: Other GTs (“QA-3-O-TriS sequences”) that can be used in QA glycosylation

[0427]

[0428] Table 7: Auxiliary Activity

[0429]

[0430] Table 8: QA Biosynthetic Activity

[0431]

[0432] Table 9: DNA (top right) and protein (bottom left) sequence identity between the gene and protein sequences of the three UGT sequences identified in the *Sapindus mukorossi* genome, the novel sequence DN20529_c0_g2_i8 identified from the de novo transcriptome, and Qs-3-O-RhaT / XylT. The Qs_0283860 region corresponds to the genomic region of the Qs_0283860 pseudogene between the predicted start codon and the predicted stop codon.

[0433]

[0434]

[0435] Table 10: Primers used for cloning four glycosyltransferases. Gene-specific sequences are shown in black. The attB site required for cloning is shown in gray.

[0436]

[0437] serial number <![CDATA[δ C Type <![CDATA[δ H mult(J, in Hz)]]> serial number <![CDATA[δ C Type <![CDATA[δ H mult(J, in Hz)]]> 1 <![CDATA[39.4,CH2]]> 1.70,d(13.3) / 1.13,m 19 <![CDATA[47.8,CH2]]> 2.30 / 1.04,m 2 <![CDATA[25.8,CH2]]> 2.03 / 1.78,m 20 31.6,Cq - 3 83.0,CH 3.94,dd(12,4.4) 21 <![CDATA[36.7,CH2]]> 1.96 / 1.15,m 4 56.4,Cq - 22 <![CDATA[32.9,CH2]]> 1.91 / 1.77,m 5 49.1,CH 1.35,m 23 209.3,CH 9.42,s 6 <![CDATA[21.6,CH2]]> 1.50 / 0.90,m 24 <![CDATA[10.5,CH3]]> 1.11,s 7 <![CDATA[33.7,CH2]]> 1.58 / 1.26,m 25 <![CDATA[16.3,CH3]]> 1.01,s 8 41.1,Cq - 26 <![CDATA[17.9,CH3]]> 0.80,s 9 48.2,CH 1.77,m 27 <![CDATA[27.4,CH3]]> 1.40,s 10 37.2,Cq - 28 181.3,Cq - 11 <![CDATA[24.6,CH2]]> 1.94 / 1.94,m 29 <![CDATA[33.6,CH3]]> 0.89,s 12 123.3,CH 5.31,t(3.3) 30 <![CDATA[25.0,CH3]]> 0.97,s 13 145.3,Cq - GlcA-1 104.8, CH 4.20,d(7) 14 42.9,Cq - GlcA-2 75.3, CH 3.11,t(8.3) 15 <![CDATA[36.3,CH2]]> 1.84 / 1.34,m GlcA-3 77.9, CH 3.32, overlapping with methanol 16 75.4, CH 4.45,t(3.5) GlcA-4 73.7, CH 3.42,m 17 50.0,Cq - GlcA-5 76.6,CH 3.57,br s 18 42.2,CH 3.01,dd(14.0,4.3) GlcA-6 No observations were made. -

[0438] Table 11: Full NMR data (600, 150 MHz) of saponin acid 3-O-β-D-glucopyranoside (QsbAS / QsCYP716-C-16α / QsCYP714-C-23 / QsCYP716-C-28 / QsCSL1 products) in MeOH-d4.

[0439]

[0440]

[0441] Table 12: Full NMR data (600, 150 MHz) of saponin acid 3-O-β-D-glucopyranoside (QsbAS / QsCYP716-C-16α / QsCYP714-C-23 / QsCYP716-C-28 / CslG2 products) in MeOH-d4.

[0442]

[0443]

[0444] Table 13: Full NMR data (600, 150 MHz) of saponin acid 3-O-{-β-D-galactopyranosyl-(1→2)-β-D-glucopyranoside} (QsbAS / QsCYP716-C-16α / QsCYP714-C-23 / QsCYP716-C-28 / QsCSL1 / Qs-3-O-GalT products) in MeOH-d4.

[0445]

[0446]

[0447] Table 14: QA-GlcpA-[Galp]-Rhap (QsbAS / QsCYP716-C-16α / QsCYP714-C-23 / QsCYP716-C-28 / QsCslG2 / Qs-3-O-GalT / Qs_0283850 product) in MeOH-d4 1 H, 13 C NMR spectral data (400, 100MHz)

[0448]

[0449]

[0450] Table 15: QA-GlcpA-[Galp]-Xylp (QsbAS / QsCYP716-C-16α / QsCYP714-C-23 / QsCYP716-C-28 / QsCslG2 / Qs-3-O-GalT / Qs_0283870 product) in MeOH-d4 1 H, 13 C NMR spectral data (400, 100MHz)

[0451]

[0452]

[0453]

[0454] References in Table 3

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[0515] Zhao, X., Wang, P., Li, M., Jiang,

[0516] sequence

[0517] SEQ ID NO:1 - Encoding sequence (2142 bp) of saponin acid 3-O-glucuronyl transferase (cellulose synthase-like enzyme QsCSL1)

[0518]

[0519] SEQ ID NO:2 - Sequence of nucleotide translation of saponin acid 3-O-glucuronyl transferase (cellulose synthase-like enzyme QsCSL1) (713aa):

[0520] *

[0521] SEQ ID NO:3-Code sequence (1479bp) for soapberry QA-GlcpAβ-1,2-D-galactosyltransferase (Qs-3-O-GalT)

[0522]

[0523] SEQ ID NO:4 - The nucleotide translation sequence (492aa) of soapberry QA-GlcpAβ-1,2-D-galactosyltransferase (Qs-3-O-GalT):

[0524] MVESPADHDVLKIIVLPWVTSGHMIPMVDAARLFAMHGADVTIITTPANALTFQKSVDRDFNSGRLIRTHTLKFPAAEVGVPEGVENFNNTSPEMTSKVYLGVSMLREPTQQLIEDLRPDCLI TDMFYPWAVDVADKLGIPRLIFQGPGSFGLSAMHSIKQYEPFKSVTSDTETFPLPGLPHKVEMTRLQIPKWVREPNGYTQLMGRVKDSERRSYGSLVNSFYDFEGPYEEHYRKATGQRVWSIG PVSVWVNQDAADKVGRGQDLVAEDQNSWLNWLNSKEKNSVLYVSFGSMAKFPSAQLLEIAHGLEASGHSFIWVVRKVDGDDDVDVWLPDFEKKMKENNKGFIIRNWAPQLLILDHPAIGGLLN HSGWNSVLEGATAGLPMITWPLYAEHFYNERLVLDVLKIGVPVGVKEWKNLHEVGELVRRDAIAKAIKLLMGSGEEAEVMRKKAKELGVGAKKGIQVGGSSHTNLIAVIDELKSLKKSRIQGV*

[0525] SEQ ID NO:5 - Coding sequence of soapberry QA-GlcpA-Galp dual β-1,3-D-xylosyltransferase / α-1,3-L-rhamnosyltransferase (Qs-3-O-RhaT / XylT) (1515bp)

[0526]

[0527] SEQ ID NO:6 - The nucleotide translation sequence (504aa) of the soapberry QA-GlcpA-Galp dual β-1,3-D-xylosyltransferase / α-1,3-L-rhamnosyltransferase (Qs-3-O-RhaT / XylT):

[0528] MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASGYHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIV ADMFTSWTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPDGTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLI HRNDEDKIQRSHKTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDEDNDDVGNKWLPAFEERIKKENKGMILRGWAPQMLILNHPAIGGFMTHCG WNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKAAQDHES*

[0529] No. 2 part – Other biosynthetic enzymes:

[0530] SEQ ID NO:7-AsHMGR (codase encoding HMG-CoA from rough oats) (1689bp):

[0531] The full-length HMGR sequence is provided below. The 5' region (underlined) can be removed to generate a truncated, feedback-insensitive form (tHMGR). The tHMGR sequence is also given separately below.

[0532] ATGGCTGTGGAGGTTCACCGCCGGGCTCCCGCGCCCCATGGCCGGGGCACCGGGGAGAAGGGCCGCGTG CAGGCCGGGGACGCGCTGCCGCTGCCGATCCGCCACACCAACCTCATCTTCTCGGCGCTCTTCGCCGCCTCCCTCGC ATACCTCATGCGCCGCTGGAGGGAGAAGATCCGCAACTCCACGCCGCTCCACGTCGTGGGGCTCACCGAGATCTTCG CCATCTGCGGCCTCGTCGCCTCCCTCATCTACCTCCTCAGCTTCTTCGGCATCGCCTTCGTGCAGTCCGTCGTATCC AACAGCGACGACGAGGACGAGGACTTCCTCATCGCGGCTGCAGCATCCCAGGCCCCCCCGCCGCCCTCCTCCAAGCC CGCGCCGCAGCAGTGCGCCCTGCTGCAGAGCGCCGGAGTC

[0533] The nucleotide translation sequence (562aa) of SEQ ID NO:8-AsHMGR (rough oat HMG-CoA reductase):

[0534] MAVEVHRRAPAPHGRGTGEKGRVQAGDALPLPIRHTNLIFSALFAASLAYLMRRWREKIRNSTPLHVV GLTEIFAICGLVASLIYLLSFFGIAFVQSVVSNSDDEDEDFLIAAAASQAPPPPSSKPAPQQCALLQSAGV APEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDGFDYDSILGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCLIASTNRGCKAIAESGGASSVVYRDGMTRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFTCSTGDAMGMNMVSKGVQNVLD YLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSVVCEAVIREEVVHKVLKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVESS QCITMLEAVNDGRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVLAGELSLISAQAAGHLVQSHMKYNRSSKDMSKIAC*

[0535] SEQ ID NO:9-AstHMGR (1275bp encoding sequence of truncated HMG-CoA reductase from rough oats):

[0536]

[0537] The nucleotide translation sequence (424aa) of SEQ ID NO:10-AstHMGR (truncated HMG-CoA reductase from rough oats):

[0538] MAPEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDGFDYDSILGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCL IASTNRGCKAIAESGGASSVVYRDGMTRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFTCSTGDAMGMNMVSKGVQNVL DYLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSVVCEAVIREEVVHKVLKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVES SQCITMLEAVNDGRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVLAGELSLISAQAAGHLVQSHMKYNRSSKDMSKIAC

[0539] SEQ ID NO:11-Saponin β-amyrin synthase, QsbAS(OQHZ-2074321) coding sequence (2277bp):

[0540]

[0541] SEQ ID NO: 12 - A sequence translated from nucleotides of QsbAS (OQHZ - 2074321) (758 aa):

[0542] MWRLKIAEGGSDPYLFSTNNFVGRQTWEFEPEAGTPEERAEVEAARQNFYNNRYQVKPCDDLLWRYQFLREKNFKQTIPPVKVEDGQEITYEMATTSMQRAARHLSALQASDGHWPAQIAGPLFFMPPLVFCVYITGHLNTVFPSEHRKEILRYMYYHQNEDGGWGLHIEGHSTMFCTALNYICMRILGEGPEGGQDNACARARMWILDHGGVTHIPSWGKTWLSILGLFEWSGSNPMPPEFWILPSFLPMHPAKMWCYCRMVYMPMSYLYGKRFVGPITPLIVQLREEIHTQNYHEINWKSVRHLCAKEDIYYPHPLIQDLIWDSLYILTEPLLTRWPLNKLVRERALQVTMKHIHYEDENSRYITIGCVEKVLCMLACWVDDPNGDAFKKHLARVPDYVWVSEDGITMQSFGSQEWDAGFAVQALLASNLTEELGPALAKGHDFIKQSQVKDNPSGDFKSMYRHISRGSWTFSDQDHGWQVSDCTAEGLKCCLLLSMLPPEIVGEKMEPQRLFDSVNVLLSLQSKKGGLAAWEPAGAQDWLELLNPTEFFADIVVEHEYVECTGSAIQALVLFKKLYPGHRKKEIDSFITNAVRFLENTQTADGSWYGNWGVCFTYGCWFALGGLAAAGKTYNNCPAIRKAVNFLLTTQREDGGWGESYLSSPKKIYVPLEGSRSNVVHTAWAMMGLIHAGQAERDSTPLHRAAKLIINYQLENGDWPQQEITGVFMKNCMLHYPMYRNIYPMWALAEYRRRVPLP*

[0543] SEQ ID NO: 13 - QsCYP716 - C - 28 (OQHZ - 2073932) (C - 28 oxidase, previously named CYP716A224

[24] ) coding sequence (1443 bp):

[0544]

[0545] The nucleotide translation sequence (480aa) of SEQ ID NO:14-QsCYP716-C-28(OQHZ-2073932):

[0546] MEHLYLSLVLLFVSSISLSLFLFYKHKSMFTGANLPPGKIGYPLIGESLEFLSTGWKGHPEKFIFDRMSKYSSQIFKTSILGEPTAVFPGAVCNKFLFSNENKLVNAWWPASVDKIFPS SLQTSSKEEAKKMRKLLPQFLKPEALHRYIGIMDSIAQRHFADSWENKNQVIVFPLAKRYTFWLACRLFISVEDPTHVSRFADPFQLLAAGIISIPIDLPGTPFRKAINASQFIRKELLA IIRQRKIDLGEGKASPTQDILSHMLLTCDENGQYMNELDIADKILGLLVGGHDTASAACTFIVKFLAELPHIYEQVYKEQMEIAKSKVPGELLNWEDIQKMKYSWNVACEVMRLAPPLQG AFREAITDFVFNGFSIPKGWKLYWSANSTHKSPDYFPEPDKFDPTRFEGNGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKLVPDEKIVVDPMPIPAKGLPVRLYPHKA*

[0547] SEQ ID NO:15-QsCYP716-C-16α(OQHZ-2012090) (C-16α oxidase) coding sequence (1506bp / 1443bp):

[0548] The difference between the long and short subtypes, as described in this article, lies in the presence of the first 63 nucleotides (21 amino acids) in the sequence below, which are underlined.

[0549] ATGATATATAATAATGATAGTAATGATAATGAATTAGTAATCAGCTCAGTTCAGCAACCATCC

[0550] SEQ ID NO: 16 - A sequence translated from the QsCYP716 - C - 16α nucleotide (501aa / 480aa):

[0551] MIYNNDSNDNELVISSVQQPS MDPFFIFGLLLLALFLSVSFLLYLSRRAYASLPNPPPGKLGFPVVGESLEFLSTRRKGVPEKFVFDRMAKYCRDVFKTSILGATTAVMCGTAGNKFLFSNEKKHVTGWWPKSVELIFPTSLEKSSNEESIMMKQFLPNFLKPEPLQKYIPVMDIITQRHFNTSWEGRNVVKVFPTAAEFTTLLACRVFLSVEDPIEVAKISEPFEILAAGFLSIPINLPGTKLNKAVKAADQIRDAIVQILKRRRVEIAENKANGMQDIASMLLTTPTNAGFYMTEAHISEKILGMIVGGRDTASTVITFIIKYLAENPEIYNKVYEEQMEVVKSKKPGELLNWEDVQKMKYSWCVACEAMRLAPPVQGGFKVAINDFVYSGFNIRKGWKLYWSAIATHMNPEYFPEPEKFNPSRFEGKGPVPYSFVPFGGGPRMCPGKEYSRLETLVFMHHLVTRYNWEKVYPTEKITVDPMPFPVNGLPIRLIPHKHQ*

[0552] SEQ ID NO: 17 - The coding sequence (1524bp) of QsCYP714 - C - 23 (C - 23 oxidase):

[0553]

[0554] SEQ ID NO: 18 - Amino acid sequence translated from the QsCYP714 - C - 23 nucleotide sequence (507 aa):

[0555] MWFTVGLVLVFALFIRLYSSLWLKPRATRIKLSNQGIKGPKPAFLLGNVAEMRRFQSKLPKSELKQGQVSHDWASKSLFPFFSLWSQKYGNTFVFSLGNIQVLYVSDHELVKEINQNTSLDLGKPKYLQKERGPLLGQGILTSNGQLWAYQRKIMTPELYKEKIKGMCELMVESVAWLVEEWGTKIQAEGGAADIRIDEDLRSFSGDVISKACFGSCYAGGREIFLRLRALQHQIASKALLMGFPGLKYLPIKSNREIWRLEKEIFQLIMKLAEDRKKEQHERDLLQIIIEGAKSSDLSSEAMAKFIVDNCKNVYLAGHETTAMSAGWTLLLLANHPEWQARVRDEILQVTEGRNPDFDMLHKMKLLTMVIQEALRLYPTVIFMSREALEDINVGNIQVPKGVNIWIPVVNLQRDTTVWGADANEFNPERFANGVNNSCKVPQLYLPFGAGPRICPGINLAMTEIKILLCILLTKFSFSVSPNYRHSPVFKLVLEPENGINVIMKKL*

[0556] SEQ ID NO: 19 – Coding sequence of GmSGT2 (GmUGT73P2) (β - D - galactosyltransferase of Glycine max / soybean) (1488 bp):

[0557]

[0558] SEQ ID NO:20-GmSGT2(GmUGT73P2) (Sequence of nucleotide translation of soybean (Glycine max / soybean) β-D-galactosyltransferase) (495aa):

[0559] MEKKKGELKSIFLPFLSTSHIIPLVDMARLFALHDVDVTIITTAHNATVFQKSIDLDASRGRPIRTHVVNFPAAQVGLPVGIEAFNVDTPREMTPRIYMGLSLLQQVFEKLFHDLQPDFIVTDM FHPWSVDAAAKLGIPRIMFHGASYLARSAAHSVEQYAPHLEAKFDTDKFVLPGLPDNLEMTRLQLPDWLRSPNQYTELMRTIKQSEKKSYGSLFNSFYDLESAYYEHYKSIMGTKSWGIGPVSL WANQDAQDKAARGYAKEEEEKEGWLKWLNSKAESSVLYVSFGSINKFPYSQLVEIARALEDSGHDFIWVVRKNDGGEGDNFLEEFEKRMKESNKGYLIWGWAPQLLILENPAIGGLVTHCGWNT VVESVNAGLPMATWPLFAEHFFNEKLVVDVLKIGVPVGAKEWRNWNEFGSEVVKREEIGNAIASLMSEEEEDGGMRKRAKELSVAAKSAIKVGGSSHNNMKELIRELKEIKLSKEAQETAPNP*

[0560] SEQ ID NO:21-AsSQS (coarse oat squalene synthase) coding sequence (1212bp):

[0561]

[0562] The nucleotide translation sequence (403aa) of SEQ ID NO:22-AsSQS (rough oat squalene synthase):

[0563] MGALSRPEEVVALVKLRVAAGQIKRQIPAEEHWAFAYDMLQKVSRSFALVIQQLGPELRNAVCIFYLVLRALDTVEDDTSIPNDVKLPILRDFYRHVYNPD WRYSCGTNHYKVLMDKFRLVSTAFLELGEGYQKAIEEITRRMGAGMAKFICQEVETIDDYNEYCHYVAGLVGYGLSRLFHAAGTEDLASDQLSNSMGLFLQ KTNIIRDYLEDINEIPKCRMFWPREIWSKYADKLEDLKYEENSEKAVQCLNDMVTNALVHAEDCLQYMSALKDNTNFRFCAIPQIMAIGTCAICYNNVKVFRGVVKMRRGLTARIIDETKSMSDVYSAFYEFSSLLESKIDDNDPSSALTRKRVEAIKRTCKSSGLLKRRGYDLEKSKYRHMLIMLALLLVAIIFGVLYAK*

[0564] SEQ ID NO:23-AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2) coding sequence (2325bp):

[0565]

[0566] SEQ ID NO:24 - The nucleotide translation sequence (774aa) of AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2):

[0567] *

[0568] SEQ ID NO:25 - Encoding sequence (2124bp) of soapberry saponin acid 3-O-glucuronyl transferase (cellulose synthase-like enzyme QsCslG2):

[0569]

[0570] SEQ ID NO:26 - Sequence of nucleotide translation of soapberry saponin 3-O-glucuronyl transferase (cellulose synthase-like enzyme QsCslG2) (707aa):

[0571] MATVSSLHTCTVQQPRAAINRIHIFLHFIAILFLFYYRVTGLFYDNAVPTLAWSLMTLAELIFAFVWVLSQAFRWRPVLRSVIPERIPKDVRLPAVDILICTADPLKEPTVEVMNTVLSAMALDYPAENLAVYLSDDGGSPVTLFAMKQVGPFAKLWLPFCNKYGIKTRHPESFFS AFADDERLHRSDEFRAEEEAIKDKYEEFKRTIEKYGGEGKNSHVVQDRPPHVEIIHDTRKIRENSEDQAVPLLVYVSREKRPSYNSRFKAGALNTLLRVSGVISNSPYVLVLDCDMYCNDPTSARQAMCFHLDPQMSRTLSFVQFPQVFYNVSKNDIYDGQARAAFKTKWQGMDGLR GPLLSGTGFYLKRKSLYGSPNQEDDCLLEPHKNFGKCDKLIESVKVIYERDVSIKADSSDAILQDAKQLASCPYETNTSWGKEVGFSYDCLLESTFTGYLLHCRGWTSVYLYPKKPCFLGCTPVDMKEAMVQYTKWISELFLLAISRFNPLTFGISRMSILQSMCYGYLTIMPILSV AMIFYATVPQLCLLRGVPLFPKVSDPWFAVFLAIFVSSLCQHLIEVLTSDGTLKTWWNEQRNWVIKSGSGSVFGALSGILKWFGMKIKFGLSNKAVDKEKLEKYEKGKFDFQGAAMFMVPLTISVILNTLCLIGGLWRVITLKNFEEMSGQFIISLYFLALSYPILEGLLRKGKGKA

[0572] SEQ ID NO:27 - Coding sequence (1485bp) of soapberry QA-GlcpA-Galpα-1,3-L-rhamnosyltransferase (Qs_0283850):

[0573]

[0574] SEQ ID NO:28 - The nucleotide translation sequence (494aa) of soapberry QA-GlcpA-Galpα-1,3-L-rhamnosyltransferase (Qs_0283850):

[0575] MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKGKTSGYHVNIHAVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPD CIVADMFNRWTSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDGAFSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGP LQLIYQNDDPKVQRSQKTAVVSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVGNKWLPDFEERIKRENKGIIFRGWAPQMLILNHPAIGGFMT HCGWNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDCGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKVA

[0576] SEQ ID NO:29-coding sequence (1491bp) of soapberry QA-GlcpA-Galpα-1,3-L-rhamnosyltransferase (TRINITY_DN20529_c0_g2_i8):

[0577]

[0578] SEQ ID NO:30 - The nucleotide translation sequence (496aa) of soapberry QA-GlcpA-Galpα-1,3-L-rhamnosyltransferase (TRINITY_DN20529_c0_g2_i8):

[0579] MVSGDDTVSRPLIVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKRKNSGYHVNIHTVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPDCI VADMFNRWTSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDGAFSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGPLQ LIYQNDDPKVQRSQKTAVLSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVGNKWLPGFEERIKRENKGIIFRGWAPQMLILNHPAIGGFMTHCG WNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKAAMKS

[0580] SEQ ID NO:31 - Coding sequence (1515bp) of soapberry QA-GlcpA-Galpβ-1,3-D-xylosyltransferase (Qs_0283870):

[0581]

[0582] AGAAGATATTAGACGGTGGAGAAGAGGCGGAAGGAATGAGAAAGAGGGCCCGGGAGCTGAAAGAAATAGCTAAAAGAAGTGTGGAAGAAGGTGGGTCCTCTTATAATAATTTAACGGCTATGATTCAAGATCTGAAAGAATTTAGAGCTAACAATGGCAAGGCTGCACAAGATCATGAATCGTGA

[0583] SEQ ID NO:32 - A sequence translated from nucleotides of Quillaja saponaria QA - GlcpA - Galpβ - 1,3 - D - xylosyltransferase (Qs_0283870) (504aa):

[0584] MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASGYHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIVADMFTSWTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPDGTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLIHRNDEDKIQRSHKTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDEDNDDVGNKWLPAFEERIKKENKGMILRGWAPQMLILNHPAIGGFMTHCGWNAAVEALSSGVPIITFPVFSDQFYNERLISQVHKCGVGVGTEAWSYAFDAGKNPVGREKIMTAVKKILDGGEEAEGMRKRARELKEIAKRSVEEGGSSYNNLTAMIQDLKEFRANNGKAAQDHES

Claims

1. A method of converting a host, which is a plant or a yeast, from a host phenotype which is not capable of performing 3-O branched trisaccharide quillaic acid QA-3-O-TriS biosynthesis into a host phenotype which is capable of performing said QA-3-O-TriS biosynthesis by glycosylation of quillaic acid (QA) at its 3-O position, said QA-3-O-TriS is 3β-{[β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucuronopyranoside]oxy}-quillaic acid (QA-GlcpA-[Galp]-Xylp) or (3β-{[α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucuronopyranoside]oxy}-quillaic acid) (QA-GlcpA-[Galp]-Rhap), said method comprising, after the step of introducing a heterologous nucleic acid into said host, the step of expressing said heterologous nucleic acid in said host or one or more cells thereof, wherein said heterologous nucleic acid comprises a plurality of nucleotide sequences, each of said plurality of nucleotide sequences encoding a polypeptide which in combination has said QA-3-O-TriS biosynthetic activity, wherein said heterologous nucleic acid encodes polypeptides of the following types (i), (ii) and (iii): (i) a QA 3-O glucuronosyltransferase "QA-GlcAT" which is capable of transferring a D-glucuronic acid "GlcpA" to the 3-O position of quillaic acid to form 3β-{[β-D-glucuronopyranoside]oxy}-quillaic acid "QA-GlcpA"; (ii) a QA-GlcpA galactosyltransferase "QA-GalT" which is capable of transferring a D-galactose "Galp" via a β-1->2 linkage to QA-GlcpA to form 3β-{[β-D-galactopyranosyl-(1->2)-β-D-glucuronopyranoside]oxy}-quillaic acid "QA-GlcpA-Galp"; (iii) a QA-GlcpA-Galp rhamnosyl / xylosyltransferase "QA-RhaT / XylT" which is capable of transferring a L-rhamnose "Rhap" and / or a 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-glucuronopyranoside]oxy}-quillaic acid) "QA-GlcpA-[Galp]-Rhap" and / or 3β-{[β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucuronopyranoside]oxy}-quillaic acid "QA-GlcpA-[Galp]-Xylp"; wherein each polypeptide is selected from the list: (i) a QA-GlcAT as depicted in SEQ ID: No 2 or 26; (ii) a QA-GalT as depicted in SEQ ID: No 4; (iii) a QA-RhaT / XylT as depicted in SEQ ID: No 6 or 30. (iii) QA-RhaT / XylT as shown in SEQ ID: No 6, 28, 30 or 32.

2. The method of claim 1, wherein the heterologous nucleic acid further comprises a plurality of nucleotide sequences each encoding a polypeptide having QA biosynthetic activity in combination, "QA polypeptide", wherein the nucleic acid encodes all of the following QA polypeptides: (i) a beta-amyrin synthase "bAS" as shown in SEQ ID: No 12 for cyclizing 2,3-oxidosqualene (OS) into a triterpene; (ii) an enzyme "C-28 oxidase" as shown in SEQ ID: No 14 capable of oxidizing beta-amyrin at the C-28 position into a carboxylic acid; (iii) an enzyme "C-16a oxidase" as shown in SEQ ID: No 16 capable of oxidizing beta-amyrin at the C-16a position into an alcohol; and (iv) an enzyme "C-23 oxidase" as shown in SEQ ID: No 18 capable of oxidizing beta-amyrin at the C-23 position into an aldehyde.

3. The method of claim 1, wherein the nucleic acid further comprises a plurality of nucleotide sequences encoding one or more of the following polypeptides: (i) HMG-CoA reductase (HMGR); (ii) squalene synthase (SQS) wherein the HMGR is selected from polypeptides SEQ ID NO 8 and 10, and SQS is selected from polypeptides SEQ ID NO 22 and 24.

4. The method of claim 3, wherein the HMGR is encoded by a polynucleotide sequence having SEQ ID NO 7 or 9, and SQS is encoded by a polynucleotide sequence having SEQ ID NO 21 or 23.

5. The method of claim 1, wherein the nucleotide sequences are present on two or more different nucleic acid molecules.

6. The method of claim 5, 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.

7. The method of claim 6, wherein the nucleic acid molecules are transient expression vectors.

8. The method of claim 7, wherein each of the transient expression vectors comprises an expression cassette comprising: (i) a promoter operably linked to (ii) an enhancer sequence from an RNA-2 genome segment of a bipartite RNA virus, wherein a target initiation site in the RNA-2 genome segment has been mutated; (iii) a nucleotide sequence encoding one of the polypeptides having the QA-3-O-TriS biosynthetic activity in combination; and (iv) a terminator sequence.

9. The method of claim 8, wherein the expression cassette further comprises (v) a 3' UTR upstream of the terminator sequence.

10. The method of claim 1, wherein the host is a plant.

11. A yeast host cell, which host cell contains or is transformed with a heterologous nucleic acid, which heterologous nucleic acid comprises a plurality of nucleotide sequences, each of which encodes a polypeptide having QA-3-O-TriS biosynthetic activity in combination, wherein expression of the nucleic acid confers on the transformed host the ability to carry out QA-3-O-TriS biosynthesis, wherein the nucleic acid encodes polypeptides of the following types (i), (ii) and (iii): (i) a QA-GlcAT capable of transferring GlcpA to the 3-O position of a saponic acid to form QA-GlcpA; (ii) a QA-GalT capable of transferring Galp via a beta-1->2 linkage to QA-GlcpA to form QA-GlcpA-Galp; (iii) a QA-RhaT / XylT capable of transferring Rhap and / or Xylp via a 1,3 linkage to QA-GlcpA-Galp to form QA-GlcpA-[Galp]-Rhap and / or QA-GlcpA-[Galp]-Xylp, respectively; wherein each polypeptide is selected from the list: (i) a QA-GlcAT as set forth in SEQ ID: No 2 or 26; (ii) a QA-GalT as set forth in SEQ ID: No 4; (iii) a QA-RhaT / XylT as set forth in SEQ ID: No 6, 28, 30 or 32.

12. The yeast host cell according to claim 11, wherein the host cell is obtainable by the method according to any one of claims 1 to 5.

13. The yeast host cell according to claim 11 or 12, which host cell contains or is transformed with a heterologous nucleic acid, which heterologous nucleic acid further comprises a plurality of nucleotide sequences each encoding a polypeptide having QA biosynthetic activity in combination, wherein the nucleic acid encodes all of the following QA polypeptides: (i) a beta-amyrin synthase "bAS" as set forth in SEQ ID: No 12 for cyclizing 2,3-oxidosqualene (OS) to a triterpene; (ii) an enzyme "C-28 oxidase" as set forth in SEQ ID: No 14 capable of oxidizing beta-amyrin at the C-28 position to a carboxylic acid; (iii) an enzyme "C-16a oxidase" as set forth in SEQ ID: No 16 capable of oxidizing beta-amyrin at the C-16a position to an alcohol; and (iv) an enzyme "C-23 oxidase" as set forth in SEQ ID: No 18 capable of oxidizing beta-amyrin at the C-23 position to an aldehyde.

14. A method for producing a host cell according to any one of claims 11 to 13 by co- permeabilizing a plurality of recombinant constructs comprising a heterologous nucleic acid as defined in any one of claims 11 to 13 into the cell for transient expression of the nucleic acid.

15. A method for producing a host cell according to any one of claims 11 to 13 by transforming a cell with a heterologous nucleic acid by introducing the 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.

16. A recombinant plant or yeast vector comprising a QA-3-O-TriS biosynthesis nucleotide sequence encoding the following types of polypeptides (i), (ii) and (iii): (i) a QA 3-O glucuronosyltransferase "QA-GlcAT" capable of transferring a D-glucuronic acid "GlcpA" to the 3-O position of a quillaic acid to form 3 -{[ -D-glucopyranosyluronic acid]oxy}- quillaic acid "QA-GlcpA"; (ii) a QA-GlcpA galactosyltransferase "QA-GalT" capable of transferring a D-galactose "Galp" via a -1 -> 2 linkage to QA-GlcpA to form 3 -{[ -D-galactopyranosyl-(1 -> 2)- -D-glucopyranosyluronic acid]oxy}-quillaic acid "QA-GlcpA-Galp"; (iii) a QA-GlcpA-Galp rhamnosyl / xylosyltransferase "QA-RhaT / XylT" capable of transferring a L-rhamnose "Rhap" and / or a D-xylose "Xylp" via a 1,3 linkage to QA-GlcpA-Galp to form (3 -{[ -L-rhamnopyranosyl-(1 -> 3)-[ -D-galactopyranosyl-(1 -> 2)]- -D-glucopyranosyluronic acid]oxy}-quillaic acid) "QA-GlcpA-[Galp]-Rhap" and / or 3 -{[ -D-xylopyranosyl-(1 -> 3)-[ -D-galactopyranosyl-(1 -> 2)]- -D-glucopyranosyluronic acid]oxy}-quillaic acid "QA-GlcpA-[Galp]-Xylp"; wherein each polypeptide is selected from the list: (i) a QA-GlcAT as set forth in SEQ ID: No 2 or 26; (ii) a QA-GalT as set forth in SEQ ID: No 4; (iii) a QA-RhaT / XylT as set forth in SEQ ID: No 6, 28, 30 or 32; wherein the nucleotide sequence is operably linked to a promoter for transcription in a host cell.

17. The vector according to claim 16, wherein each QA-3-O-TriS biosynthesis nucleotide sequence is selected from SEQ ID NO: 1, 3, 5, 25, 27, 29 or 31.

18. The vector according to claim 16 or 17, wherein the promoter is an inducible promoter.

19. The vector according to claim 16 or 17, which is a plant vector.

20. The vector according to claim 19, wherein the vector comprises an expression cassette comprising: (i) a promoter operably linked to (ii) a QA-3-O-TriS biosynthesis nucleotide sequence as set forth in SEQ ID NO: 1, 3, 5, 25, 27, 29 or 31; and (iii) a terminator. (ii) an enhancer sequence from an RNA-2 genome segment of a bipartite RNA virus, wherein the target initiation site in the RNA-2 genome segment has been mutated; (iii) the QA-3-O-TriS biosynthesis nucleotide sequence; and (iv) a terminator sequence.

21. The vector of claim 20, wherein the expression cassette further comprises (v) a 3'UTR upstream of the terminator sequence.

22. A method of transforming a host cell, the method comprising the step of introducing one or more different vectors according to any one of claims 16 to 18 into a plant or yeast host cell to transform the host cell.

23. The method of claim 22, wherein the one or more different vectors collectively encode: (i) a QA-GlcAT capable of transferring GlcpA to the 3-O position of saponic acid to form QA-GlcpA; (ii) a QA-GalT capable of transferring Galp via a beta-1->2 linkage to QA-GlcpA to form QA-GlcpA-Galp; (iii) a QA-RhaT / XylT capable of transferring Rhap and / or Xylp via a 1,3 linkage to QA-GlcpA-Galp to form QA-GlcpA-[Galp]-Rhap and / or QA-GlcpA-[Galp]-Xylp, respectively; wherein each polypeptide is selected from the list: (i) a QA-GlcAT as set forth in SEQ ID: No 2 or 26; (ii) a QA-GalT as set forth in SEQ ID: No 4; (iii) a QA-RhaT / XylT as set forth in SEQ ID: No 6, 28, 30 or 32.

24. A yeast host cell, the host cell containing or transformed with a vector according to any one of claims 16 to 18.

25. The yeast host cell of claim 24, the host cell further containing or transformed with a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide, the polypeptide being a plant cytochrome P450 reductase (CPR) as set forth in SEQ ID No:

23.

26. A method for producing a transgenic plant, the method comprising the steps of: (a) performing the method of claim 22, wherein the host cell is a plant cell, and (b) regenerating a plant from the transformed plant cell.

27. The yeast host cell of claim 24 or 25, the host cell further comprising a heterologous nucleic acid comprising all of: (i) a beta-amyrin synthase coding sequence as set forth in SEQ ID: No 11; (ii) a C-28 oxidase coding sequence as set forth in SEQ ID: No 13; (iii) a C-16 alpha oxidase coding sequence as set forth in SEQ ID: No 15; (iv) a C-23 oxidase coding sequence as set forth in SEQ ID: No 17.

28. Use of a polypeptide encoded by the QA-3-O-TriS biosynthesis nucleotide sequence according to claim 17 to catalyze the biosynthesis of QA-3-O-TriS.

29. Use of a polypeptide according to claim 28 in a method of synthesizing QA-3-O-TriS.

30. A method for interfering with or affecting the biosynthesis of QA-3-O-TriS in a plant or yeast host, the method comprising the steps of: introducing a vector according to claim 16 or 17 into a cell of the host, followed by causing or allowing the vector to express within the cell of the host.

31. A method of producing a product, which is a glycosylated QA, in a plant or yeast host, the method comprising performing the method according to any one of claims 1 to 10, 22, 23, or 30, wherein the glycosylated QA is QA-3-O-TriS.

32. The method according to claim 31, wherein the host is a plant.

33. The method according to claim 31, further comprising separating the product from the host.

34. A method of producing a product, which is a glycosylated QA, in a heterologous plant or yeast host, the method comprising culturing a host cell according to any one of claims 11 to 13, 24 to 25, or claim 27, and purifying the product therefrom, wherein the glycosylated QA is QA-3-O-TriS.

Images