Saponin biosynthase

Abstract

In particular, the present invention relates to a method for producing triterpenes by one or more of the following: (i) a Saponia officincis [beta]-amyrin synthase (SobAS); (ii) a saponaria officinalis C28 oxidase (SoC28); (iii) a saponaria officinalis C28C16 oxidase (SoC28C16); (iv) a saponaria officinalis C23 oxidase (SoC23); (v) saponaria saponin acid 3-O glucuronic acid transferase (SoCSL); (vi) saponaria officinalis QA-GlcA (SoC3Gal); (vii) a saponaria officinalis QA-GlcA-Gal xylosyltransferase (SoC < 3 > Xyl); (viii) a saponaria officinalis QA-Tri fucosyltransferase (SoC28Fu), (viii) a saponaria officinalis QA-Tri (ix) a saponaria officinalis QA-TriF rhamnotransferase (SoC28Rha), and (x) a transferase (SoC28Rha) (x) a saponaria officinalis QA-TriFR xylosyltransferase (SoC28Xyl1); (xi) a saponaria officinalis QA-TriFRX xylosyltransferase (SoC28Xyl2); (xii) a saponaria officinalis QA-TriFRXX quinosyltransferase (SoGH1) and (xiii) a saponaria officinalis QA-TriF (Q) RXX acetyltransferase (SoBAHD1) polypeptide. In addition, various methods, host cells, isolated polypeptides, nucleic acids, and plants are also provided.

Description

Technical Field

[0001] The present invention relates to the biosynthesis of complex triterpenoid saponins such as quince acid and intermediates, and to genes and polypeptides involved in the biosynthesis. Background Art

[0002] Saponaria officinalis (Caryophyllaceae), commonly known as soapwort, is a perennial flowering plant native to Europe and Asia that has been used as a traditional soap source [1]. Saponaria officinalis is known to have detergency properties, thanks to the high levels of amphiphilic saponins in its plant extracts. The ancient Greeks, Romans, and Egyptians used saponaria extracts to clean and wash clothes. Later, the first American colonists brought saponaria plants from Europe to North America for domestic use [2]. In addition to its detergency properties, saponaria extracts have been used in folk medicine to treat syphilis, gout, rheumatism, and jaundice [3]. In addition, as an important part of Middle Eastern culture, saponaria extracts have been used in the Middle East to make tahini halvah, a traditional dessert [4]. Today, saponaria extracts are still used in cosmetics, health supplements, and herbal medicines [5]. In addition, the saponin layer of Sophora japonica extract has antifungal and immunotoxic activities, so some studies have explored its potential use as a food surfactant in bioremediation [6-9].

[0003] Soapwort is rich in saponins with various aglycone cores such as saponarioside, oleophyll, and oleophyll. The main saponins found in extracts of Soapwort are reported to be saponarioside A (SpA) and saponarioside B (SpB) [1]. SpA and SpB have similar chemical structures. Both consist of saponarioside aglycones (C30 triterpenes) modified with a branched trisaccharide at the C-3 position and a linear tetrasaccharide at the C-28 position. The first monosaccharide of the tetrasaccharide chain is β-D-fucose, which is bonded to β-D-quinovose with an attached acetyl group. The only chemical difference between SpA and SpB is the addition of β-D-xylose to the quinovose portion of the C28 sugar chain of SpA.

[0004] Interestingly, QS-21, a triterpenoid saponin found in the Chilean soap bark tree (Quillaja saponaria), is chemically strikingly similar to SpA and SpB ( Figure 1). QS-21 is a complex triterpenoid saponin synthesized by the Chilean Quillaja tree (Leguminosae). Biochemically, QS-21 consists of a C30 triterpenoid saponin backbone modified with a branched trisaccharide at C-3 and a linear tetrasaccharide at C-28. The terminal monosaccharide of the tetrasaccharide can be either β-D-semenose or β-D-xylose. In addition, the β-D-fucose in the tetrasaccharide also has a C18 acyl chain, which is glycosylated with arabinose. QS-21 is a powerful immunostimulant that can enhance antibody responses and specific T cell responses, thus having great potential as an adjuvant (Del Giudice et al., Seminars in Immunology, 2018, 39, 14-21; Marciani D.J., Trends in Pharmacological Sciences, 2018, 39, 6, 573-585). AS01 adjuvant is a liposome formulation of QS-21 and 3-O-deacylated-4'-monophosphoryl lipid A, which has been approved for use in GlaxoSmithKline's shingles vaccine Shingrix and malaria vaccine Mosquirix (Del Giudice et al., supra).

[0005] Although saponins and their intermediates have great commercial potential, their biosynthetic pathways are still largely unknown. Conceptually, the biosynthesis of saponins can be divided into two stages: (i) biosynthesis of the sapogenin nucleus; and (ii) modification of the sapogenin nucleus. Figure 2 ). However, the actual sequence in plants may be different and more details are not known. Many plant natural products are found in low abundance in plants and chemical synthesis is often not feasible due to their complex chemical structures. By understanding the biosynthetic pathways, metabolic engineering can be performed in alternative host systems to achieve large-scale production of target compounds. Summary of the invention

[0006] The inventors have identified and characterized genes involved in the biosynthesis of complex triterpenoid saponins in the Soapwort plant. Such genes include genes encoding enzymes involved in the biosynthesis of saponic acid; and genes encoding glycosyltransferases involved in the glycosylation of saponic acid. Expression of one or more of these genes can be used to produce saponic acid and saponic acid glycosylation products.

[0007] A first aspect of the present invention provides a method for producing triterpenes, comprising one or more of the following:

[0008] (i) contacting 2,3-epoxysqualene (OS) with a Saponaria officinalis β-amyrin synthase (SobAS) comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8, so that 2,3-epoxysqualene is converted into β-amyrin;

[0009] (ii) any of the following:

[0010] (a) contacting β-amyrin with a SoC28 oxidase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, so that the C-28 position of β-amyrin is oxidized to a carboxylic acid to produce oleanolic acid, and contacting oleanolic acid with a SoC28C16 oxidase polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 4, so that the C-16 position of oleanolic acid is oxidized to an alcohol to produce echinocystic acid; or

[0011] (b) contacting β-amyrin with a SoC28C16 oxidase polypeptide comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 4, so that the C-28 position of β-amyrin is oxidized to a carboxylic acid and

[0012] The C-16 position of β-amyrin is oxidized to alcohol, thereby generating echinocystic acid;

[0013] (iii) contacting the echinocystic acid with a SoC23 oxidase polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 6, such that the C-23 position of the echinocystic acid is oxidized to an aldehyde, thereby generating quince acid (QA);

[0014] (iv) contacting QA with a Soapwort QA3-O-glucuronyltransferase (SoCSL) polypeptide comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10, such that QA is converted to QA-GlcA;

[0015] (v) contacting QA-GlcA with a Saponaria officinalis QA-GlcA galactosyltransferase (SoC3Gal) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 12, such that QA-GlcA is converted to QA-GlcA-Gal;

[0016] (vi) contacting QA-GlcA-Gal with a Saponaria officinalis QA-GlcA-Gal xylosyltransferase (SoC3Xyl) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 14, such that QA-GlcA-Gal is converted to QA-Tri (QA-GlcA-Gal-Xyl);

[0017] (vii) contacting QA-Tri with a Saponaria officinalis QA-Tri fucosyltransferase (SoC28Fu) polypeptide comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 16, such that QA-Tri is converted to QA-TriF;

[0018] (viii) contacting QA-TriF with a Saponaria officinalis QA-TriF rhamnosyltransferase (SoC28Rha) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 18, such that QA-TriF is converted to QA-TriFR;

[0019] (ix) contacting QA-TriFR with a Saponaria officinalis QA-TriFR xylosyltransferase (SoC28Xyl1) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 20, such that QA-TriFR is converted to QA-TriFRX; and / or

[0020] (x) contacting QA-TriFRX with a Saponaria officinalis QA-TriFRX xylosyltransferase (SoC28Xyl2) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 22, such that QA-TriFRX is converted to QA-TriFRXX;

[0021] (xi) contacting QA-TriFRXX with a Saponaria officinalis QA-TriFRXX quinoside transferase (SoGH1) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 34, such that QA-TriFRXX is converted to QA-TriF(Q)RXX; and / or

[0022] (xii) contacting QA-TriF(Q)RXX with a Saponaria officinalis QA-TriF(Q)RXX acetyltransferase (SoBAHD1) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:36, such that QA-TriF(Q)RXX is converted to saponin B (SpB).

[0023] The method of the first aspect may include:

[0024] contacting β-amyrin with a saponaria officinalis C28 oxidase (SoC28 oxidase) to oxidize the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid,

[0025] wherein the amino acid sequence of SoC28 oxidase has at least 80% sequence identity with SEQ ID NO: 2;

[0026] contacting oleanolic acid with a saponaria officinalis C28C16 oxidase (SoC28C16 oxidase) to oxidize the C-16 position of oleanolic acid to an alcohol, thereby forming echinocystic acid,

[0027] wherein the amino acid sequence of SoC28C16 oxidase has at least 50% sequence identity with SEQ ID NO: 4; and

[0028] contacting echinocystic acid with a saponin C-23 oxidase (SoC23 oxidase) to oxidize the C-16 position of echinocystic acid to an aldehyde, thereby forming quince acid (QA),

[0029] The amino acid sequence of SoC23 oxidase has at least 50% sequence identity with SEQ ID NO:6.

[0030] The method of the first aspect may include:

[0031] contacting β-amyrin with a saponaria officinalis C28C16 oxidase (SoC28C16 oxidase) to oxidize the C-28 position of β-amyrin to a carboxylic acid and the C-16 position of β-amyrin to an alcohol, thereby forming echinococcal acid, wherein the amino acid sequence of SoC28C16 oxidase has at least 50% sequence identity with SEQ ID NO: 4; and

[0032] The echinocystic acid is contacted with a saponin C-23 oxidase (SoC23 oxidase) to oxidize the C-16 position of the echinocystic acid to an aldehyde, thereby forming quince acid (QA), wherein the amino acid sequence of the SoC23 oxidase has at least 50% sequence identity with SEQ ID NO:6.

[0033] The method of the first aspect may include or further include:

[0034] contacting QA with a Saponaria officinalis QA 3-O glucuronyltransferase (SoCSL) to covalently attach D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA or QA-Mono),

[0035] wherein the amino acid sequence of SoCSL has at least 60% sequence identity with SEQ ID NO: 10;

[0036] contacting QA-GlcA with a saponaria officinalis QA-GlcA galactosyltransferase (SoC3Gal) to covalently attach D-galactose (Gal) to QA-GlcA via a β-1→2 bond, thereby forming 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal or QA-Di),

[0037] wherein the amino acid sequence of SoC3Ga has at least 50% sequence identity with SEQ ID NO: 12; and

[0038] contacting QA-GlcA-Gal with a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) to covalently attach D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-Tri or "QA-GlcA-Gal-Xyl");

[0039] The amino acid sequence of SoC3Xyl has at least 50% sequence identity with SEQ ID NO:14.

[0040] The method of the first aspect may include or further include:

[0041] 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-Tri) is contacted with a saponin QA-Tri fucosyltransferase (SoC28Fu) to attach fucose to the 28-O position of QA-Tri, thereby forming 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF),

[0042] wherein the amino acid sequence of SoC28Fu has at least 60% sequence identity with SEQ ID NO: 16;

[0043] QA-TriF is contacted with a saponin QA-TriF rhamnosyltransferase (SoC28Rha) to covalently attach rhamnose to QA-TriF via a 1,2 bond, thereby forming 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFR),

[0044] wherein the amino acid sequence of SoC28Rha has at least 50% sequence identity with SEQ ID NO: 18;

[0045] contacting QA-TriFR with a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) to covalently attach xylose to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRX), wherein the amino acid sequence of SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; and

[0046] QA-TriFRX is contacted with a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) to covalently attach xylose to QA-TriFRX through a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 50% sequence identity to SEQ ID NO: 22.

[0047] The method of the first aspect may include or further include:

[0048] contacting QA-TriFRXX with a saponin QA-TriFRXX quinovosyltransferase (SoGH1) to covalently attach quinovose to QA-TriFRXX via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and / or

[0049] QA-TriF(Q)RXX is contacted with a Saponaria officinalis QA-TriF(Q)RXX acetyltransferase (SoBAHD1) to covalently attach an acetyl group to QA-TriF(Q)RXX, thereby forming saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO:36.

[0050] A second aspect of the present invention provides a method for converting a host from a phenotype in which the host is unable to biosynthesize triterpenes from β-amyrin to a phenotype in which the host is able to biosynthesize such triterpenes, the method comprising:

[0051] expressing a heterologous nucleic acid in said host or one or more cells thereof, preceded by a preceding step of introducing said nucleic acid into an ancestor of said host or either of said two,

[0052] Wherein, the heterologous nucleic acid encodes one, more or all of the following polypeptides:

[0053] (i) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid (SoC28 oxidase), wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2;

[0054] (ii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and the C-16 position of β-amyrin to an alcohol (SoC28C16 oxidase), the C28C16 oxidase having at least 50% sequence identity to SEQ ID NO: 4;

[0055] (iii) a SoC23 oxidase capable of oxidizing position C-23 of echinocystic acid to an aldehyde (SoC23 oxidase), the SoC23 oxidase having at least 50% sequence identity to SEQ ID NO: 6;

[0056] (iv) a Soapwort saponic acid 3-O glucuronyltransferase (SoCSL) capable of attaching D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-Mono or QA-GlcA), wherein the SoCSL has a sequence identity of at least 60% to SEQ ID NO: 10;

[0057] (v) a saponin QA-GlcA galactosyltransferase (SoC3Gal) capable of attaching D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-Di or QA-GlcA-Gal), wherein the amino acid sequence of SoC3Gal has at least 50% sequence identity to SEQ ID NO: 12;

[0058] (vi) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) capable of attaching D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 1,3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal-Xyl or QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO: 14;

[0059] (vii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) capable of attaching fucose (Fuc) to position 28-O of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), wherein the amino acid sequence of SoC28Fu has at least 50% sequence identity to SEQ ID NO: 16;

[0060] (viii) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) capable of attaching rhamnose (Rha) to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-pyranofucoidsyl ester}-saponin acid (QA-TriFR), SoC28Rha having at least 50% sequence identity to SEQ ID NO: 18;

[0061] (ix) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) capable of attaching D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of C28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20;

[0062] (x) a saponin QA-TriFRX xylosyltransferase (C28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of C28Xyl2 has at least 50% sequence identity to SEQ ID NO: 22;

[0063] (xi) a saponin QA-TriFRXX quinovose transferase (SoGH1) that attaches quinovose to QA-TrFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and / or

[0064] (xii) a Soapberry QA-TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to QA-TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO:36.

[0065] The heterologous nucleic acid in the method of the second aspect may encode the following polypeptide:

[0066] (i) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid, wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2;

[0067] (ii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and the C-16 position of β-amyrin to an alcohol (C28C16 oxidase) to form echinocystic acid, wherein the SoC28C16 oxidase has a sequence identity of at least 50% to SEQ ID NO: 4; and

[0068] (iii) a SoC23 oxidase capable of oxidizing the C-23 position of echinocystic acid to an aldehyde to form quisac acid (QA), wherein the SoC23 oxidase has a sequence identity of at least 50% to SEQ ID NO:6.

[0069] The heterologous nucleic acid in the method of the second aspect may further encode the following polypeptide:

[0070] (iv) a Saponaria officinalis QA3-O glucuronyltransferase (SoCSL) that attaches D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA), wherein the amino acid sequence of SoCSL has at least 60% sequence identity to SEQ ID NO: 10;

[0071] (v) a saponin QA-GlcA galactosyltransferase (SoC3Gal) that attaches D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-Gal), wherein the amino acid sequence of SoC3Gal has at least 50% sequence identity to SEQ ID NO: 12; and

[0072] (vi) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) that attaches D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal-Xyl or QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO: 14.

[0073] The heterologous nucleic acid in the method of the second aspect may further encode the following polypeptide:

[0074] (vii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) that attaches fucose (Fuc) to position 28-O of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), SoC28Fu having at least 60% sequence identity to SEQ ID NO: 16;

[0075] (viii) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) having a rhamnose (Rha) attached to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFR), SoC28Rha having at least 50% sequence identity to SEQ ID NO: 18;

[0076] (ix) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) that attaches D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; and

[0077] (x) a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 80% sequence identity to SEQ ID NO: 22.

[0078] The heterologous nucleic acid in the method of the second aspect may further encode the following polypeptide:

[0079] (xi) a saponin QA-TriFRXX quinovose transferase (SoGH1) that attaches quinovose (Q) to QA-TrFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and / or

[0080] (xii) a Soapberry QA-TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to QA-TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO:36.

[0081] A third aspect of the present invention provides a host cell comprising or transformed with a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a plurality of nucleotide sequences, each nucleotide sequence encoding a polypeptide, and the polypeptides in combination have triterpene biosynthetic activity,

[0082] Among them, the above-mentioned multiple nucleotide sequences encode one or more of the following polypeptides:

[0083] (i) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid (SoC28 oxidase), wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2;

[0084] (ii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and the C-16 position of β-amyrin to an alcohol (C28C16 oxidase), the C28C16 oxidase having at least 50% sequence identity to SEQ ID NO: 4;

[0085] (iii) a SoC23 oxidase capable of oxidizing position C-23 of echinocystic acid to an aldehyde (SoC23 oxidase), the SoC23 oxidase having at least 50% sequence identity to SEQ ID NO: 6;

[0086] (iv) a Sopapilla salsa 3-O glucuronyltransferase (SoCSL) that attaches D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA), wherein SoQA-GlcT has a sequence identity of at least 60% to SEQ ID NO: 10;

[0087] (v) a saponin QA-GlcA galactosyltransferase (SoC3Gal) that attaches D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-Gal), wherein the amino acid sequence of SoC3Gal has at least 50% sequence identity to SEQ ID NO: 12;

[0088] (vi) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) that attaches D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal-Xyl or QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO: 14;

[0089] (vii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) that attaches fucose (Fuc) to position 28-O of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), SoC28Fu having at least 50% sequence identity to SEQ ID NO: 16;

[0090] (viii) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) having a rhamnose (Rha) attached to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFR), SoC28Rha having at least 50% sequence identity to SEQ ID NO: 18;

[0091] (ix) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) that attaches D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20;

[0092] (x) a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 50% sequence identity to SEQ ID NO: 22;

[0093] (xi) a saponin QA-TriFRXX quinovose transferase (SoGH1) wherein quinovose (Q) is attached to QA-TriFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and / or

[0094] (xii) a Soapberry QA-TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to QA-TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO: 36,

[0095] The various nucleotide sequences in the host cell of the third aspect may encode the following polypeptides:

[0096] (i) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid, wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2;

[0097] (ii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and / or the C-16 position thereof to an alcohol (C28C16 oxidase); and

[0098] (iii) a SoC23 oxidase capable of oxidizing the C-23 position of echinocystic acid to an aldehyde to form quisac acid (QA), wherein the SoC23 oxidase has a sequence identity of at least 50% to SEQ ID NO:6.

[0099] The multiple nucleotide sequences in the host cell of the third aspect may further encode the following polypeptides:

[0100] (iv) a Sopapilla salsa 3-O glucuronyltransferase (SoCSL) that attaches D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA), wherein SoQA-GlcT has a sequence identity of at least 60% to SEQ ID NO: 10;

[0101] (v) a saponin QA-GlcA galactosyltransferase (SoC3Gal) that attaches D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-Gal), wherein the amino acid sequence of QA-GlcA-Gal has at least 50% sequence identity to SEQ ID NO: 12; and

[0102] (vi) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) that attaches D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal-Xyl, QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO: 14.

[0103] The multiple nucleotide sequences in the host cell of the third aspect may further encode the following polypeptides:

[0104] (vii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) that attaches fucose (Fuc) to position 28-O of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), SoC28Fu having at least 60% sequence identity to SEQ ID NO: 16;

[0105] (viii) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) having a rhamnose (Rha) attached to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFR), SoC28Rha having at least 50% sequence identity to SEQ ID NO: 18;

[0106] (ix) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) that attaches D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; and

[0107] (x) a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 80% sequence identity to SEQ ID NO: 22.

[0108] The multiple nucleotide sequences in the host cell of the third aspect may further encode the following polypeptides:

[0109] (xi) a saponin QA-TriFRXX quinovose transferase (SoGH1) that attaches quinovose (Q) to QA-TrFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and / or

[0110] (xii) a Soapberry QA-TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to QA-TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO:36.

[0111] The fourth aspect of the present invention provides a method for generating a host cell, comprising: transforming or transfecting the host cell with a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a plurality of nucleotide sequences as described in the second and third aspects.

[0112] A fifth aspect provides a method for producing a transgenic plant, the method comprising the following steps:

[0113] (a) performing the method of the fourth aspect; wherein the host cell is a plant cell; and

[0114] (b) regenerating a plant from the transformed plant cell.

[0115] A sixth aspect provides a transgenic plant, which can be obtained by the method of the fifth aspect, or is a clone of the transgenic plant, or a self-fertilized or hybrid progeny or other offspring,

[0116] The expression of the heterologous nucleic acid enables the transgenic plant to have a higher ability to carry out the above biosynthesis than the corresponding wild-type plant.

[0117] A seventh aspect provides a method for producing triterpenes in a heterologous host, the method comprising: culturing the host cell as described in the third aspect, and purifying the triterpenes obtained therefrom.

[0118] The eighth aspect provides a method for producing triterpenes in a heterologous host, the method comprising: growing the plant of the sixth aspect, then harvesting it, and purifying the triterpenes obtained therefrom.

[0119] The triterpene of the seventh and eighth aspects may be saponic acid or glycosylated saponic acid, such as QA-Tri, QA-TriFRXX or QA-F(Q-Ac)RXX, or an intermediate or derivative thereof.

[0120] The SobAS, SoC28 oxidase, SoC23 oxidase, SoC28C16 oxidase, SoCSL, SoC3Gal, SoC3Xyl, SoC28Fu, SoC28Rha, SoC28Xyl1, SoC28Xyl2, SoGH1 and SoBAHD1 of the first to eighth aspects may be obtained or derived from Saponaria officinalis.

[0121] Hereinafter, other aspects and embodiments of the present invention are described in more detail. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Shown is the chemical structure of saponin A / B from saponin grass ( Figure 1 A) and the chemical structure of QS-21 from Chilean Quillaja sapodilla ( Figure 1 B).

[0123] Figure 2 Shown is the putative saponin A / B biosynthetic pathway. Figure 2 (A) shows the biosynthesis of aglycone squalane from 2,3-epoxysqualene; Figure 2 (B) shows the inferred modification order of sapogenin, where QA = sapogenin; F = fucose; Rh = rhamnose; X = xylose; Q = quinovose; A = acetyl moiety; GA = glucuronic acid; Gal = galactose; Ar = arabinose. Figure 2 (C) shows the simplified chemical structures of saponin A and saponin B, where GlcA = glucuronic acid; Xyl = xylose; Gal = galactose; Fuc = fucose; Rha = rhamnose; Qui = quinovose; Acyl = acetyl moiety.

[0124] Figure 3 The expression patterns of candidate Sodexona genes in different Sodexona organs are shown, wherein the target candidate genes were identified using the Pearson correlation coefficient (PCC) between each candidate gene and SobAS1; the heat map shows the results after converting the original RNA sequence reads into regularized logarithms (rlog) and normalizing them with the size of the rlog library; the functional Sodexona genes are shown in bold; the absolute transcript reads of each candidate gene are also given in the figure.

[0125] Figure 4Shown is the characterization of SobAS, where N. benthamiana leaves transiently expressing AstHMGR and SobAS were extracted and analyzed by gas chromatography-mass spectrometry (GC / MS). Figure 4 (A) shows the total ion chromatogram (TIC) and mass spectrum, in which the extract of Nicotiana benthamiana leaves expressing only AstHMGR was used as a negative control; the peak of β-amyrin was determined by comparison with a commercial standard; Peak 1 is β-amyrin. Figure 4 (B) Shown are the functions of SobAS1.

[0126] Figure 5 Shown are the characterizations of SoCYP716A378, SoCYP716A379 and SoCYP72A984. Figure 5 (A) Shown is the pathway for the conversion of squalene to squalic acid (4) and the functions of SobAS1, SoCYP716A378, SoCYP716A379 and SoCYP72A984 in the pathway, wherein N. benthamiana leaves transiently expressing various CYP gene combinations were extracted and analyzed by GC / MS or high performance liquid chromatography mass spectrometry (HPLC / MS); extracts from N. benthamiana leaves expressing only AstHMGR were used as negative controls; peaks marked with background color are peaks identified by comparison with commercial standards (β-amyrin (1), oleanolic acid (2), echinocystic acid (3) and squalic acid (4)). Figure 5 (B) shows the GC / MS total ion chromatogram (TIC) and related mass spectra of Nicotiana benthamiana leaves transiently co-expressing tHMGR and SobAS1 via SoCYP716A378 or SoCYP716A379, wherein only one peak (2) is produced under the action of SoCYP716A378 activity, while an additional peak (3) is produced under the action of SoCYP716A379 activity. Figure 5 (C) HPLC / MS extracted ion chromatogram (EIC) and related mass spectra of Nicotiana benthamiana leaves transiently co-expressing tHMGR, SobAS1 and SoCYP716A379 with and without SoCYP72A984, wherein the EIC shown is m / z = 485.3267, i.e., sapote (4) [MH] - Calculated value of the mass of the adduct; Under the additional action of SoCYP72A984 activity, a new main peak corresponding to (4) is generated.

[0127] Figure 6 Shown is the characterization of SoCSL1. Figure 6(A) shows the structure of 3-O-{β-D-pyranoglucuronic acid}-saponin (QA-Mono, (5)) produced when SoCSL1 acts together with the soapwort enzyme required for the production of saponin (QA, (4)), wherein the background color marked part shows the modification effect of SoCSL1; the calculated values ​​of the relevant adducts and fragments of (5) are listed in the appendix. Figure 6 (B) shows a representative extracted ion chromatogram (EIC) and tandem mass spectrometry (MS / MS) spectrum of HPLC / MS analysis after extraction of Nicotiana benthamiana leaves transiently co-expressing various genes, wherein the EIC shown is m / z = 661.3588, i.e., [MH] of (5) - Calculated value of the mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379 and SoCYP72A984) required for the production of saponin (4); under the additional action of SoCSL1 activity, a peak corresponding to (5) was generated, which was determined by comparison with the standard authentic product.

[0128] Figure 7 Shown is the characterization of SoUGT73DL1. Figure 7 (A) shows the structure of 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-Di, (6)) produced when SoUGT73DL1 acts together with the saponin enzyme required for the production of 3-O-{β-D-pyranoglucuronic acid}-saponin (QA-mono, (5)), wherein the background color marked portion shows the modification effect of SoUGT73DL1; the attached table lists the calculated values ​​of the relevant adducts and fragments of (6). Figure 7 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=823.4116, i.e., [MH] - Calculated value of the mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984 and SoCSL1) required for the production of QA-mono (5); under the additional action of SoUGT73DL1 activity, a peak corresponding to (6) was generated, which was determined by comparison with the standard authentic product.

[0129] Figure 8Shown is the characterization of SoUGT73CC6. Figure 8 (A) shows the structure of 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronic acid}-saponic acid (QA-Tri, (7)) produced when SoUGT73CC6 acts together with the saponin enzyme required for the production of 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-glucuronic acid}-saponic acid (QA-Di, (6)), wherein the background color marked portion shows the modification effect of SoUGT73CC6; the calculated values ​​of the relevant adducts and fragments of (7) are listed in the appendix. Figure 8 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=955.4539, i.e., [MH] of (7) - Calculated value of the mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984, SoCSL and SoUGT73DL1) required for the production of QA-Di (6); under the additional action of SoUGT73CC6 activity, a peak corresponding to (7) was generated, which was determined by comparison with the standard authentic product.

[0130] Fig. 9 Shown are the characterizations of SoUGT74CD1 and SoSDR. Fig. 9 (A) shows the structure of 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronic acid}-saponin (QA-Tri, (7)) produced when SoUGT74CD1 reacts with the saponin enzyme required for the production of 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF, (8)), wherein the background color marked portion shows the modification effect of SoUGT74CD1; the attached table lists the calculated values ​​of the relevant adducts and fragments of (8). Fig. 9 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=1101.5118, i.e., [MH] of (8) -Calculated value of the mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984, SoCSL, SoUGT73DL1 and SoUGT73CC6) required for the production of QA-Tri (7); under the additional action of SoUGT74CD1 activity, a peak corresponding to (8) was produced, which was determined by comparison with the standard authentic; in the case of adding SoUGT74CD1, two additional small peaks (8') and (8") were also observed; in the case of adding SoSDR and SoUGT74CD1 at the same time, the yield of peak (8) was greatly increased, but in the absence of SoUGT74CD1, (8) could not be produced.

[0131] Fig.10 Shown is the characterization of SoUGT79T1. Fig.10 (A) shows the structure of 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriFR, (9)) produced when SoUGT79T1 reacts with the saponin enzyme required for the production of 3-O-{β-D-pyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF, (8)), wherein the background color marked portion shows the modification effect of SoUGT79T1; the attached table lists the calculated values ​​of the relevant adducts and fragments of (9). Fig.10 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=1247.5679, i.e., [MH] of (9) - Calculated mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984, SoCSL, SoUGT73DL1, SoUGT73CC6 and SoUGT74CD1) required for the production of QA-TriF (8); with the additional effect of SoUGT79T1 activity, a peak corresponding to (9) was generated, which was first determined based on the MS / MS spectrum and then by comparing the downstream products with the standard authentic products.

[0132] Fig.11 Shown is the characterization of SoUGT79L3. Fig.11 (A) Shown is the 3-O-{β-D-pyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1→2)-β-D-fucopyranosyl}-saponin (QA-TriFR, (9)) produced by SoUGT79L3 in combination with the saponin enzyme required for the production of 3-O-{β-D-pyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1→2)-β-D-fucopyranosyl}-saponin (QA-TriFR, (9)). )-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranoxylanosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-pyranofucosyl ester}-saponic acid (QA-TriFRX, (10)), wherein the background color marked part shows the modification effect of SoUGT79L3; the calculated values ​​of the relevant adducts and fragments of (10) are listed in the attached table. Fig.11 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=1379.6119, i.e., [MH] of (10) - Calculated mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984, SoCSL, SoUGT73DL1, SoUGT73CC6, SoUGT74CD1 and SoUGT79T1) required for the production of QA-TriFR (9); under the additional action of SoUGT79L3 activity, a peak corresponding to (10) was generated, which was first determined based on the MS / MS spectrum and then by comparing the downstream products with the standard authentic products.

[0133] Fig.12 Shown is the characterization of SoUGT73M2. Fig.12(A) Shown is the production of 3-O-{β-D-pyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRX, (10)). )-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1→3)-β-D-pyranosyl-(1→4)-α-L-pyranosyl-rhamnosyl-(1→2)-β-D-pyranoglucuronic acid}-saponic acid (QA-TriFRXX, (11)), wherein the background color marked part shows the modification effect of SoUGT73M2; the calculated values ​​of the relevant adducts and fragments of (11) are listed in the attached table. Fig.12 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=1511.6542, i.e., [MH] of (11) - Calculated mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984, SoCSL, SoUGT73DL1, SoUGT73CC6, SoUGT74CD1, SoUGT79T1 and SoUGT79L3) required for the production of QA-TriFRX (10); under the additional action of SoUGT73M2 activity, a peak corresponding to (11) was generated, which was first determined based on the MS / MS spectrum and then by comparing the downstream products with the standard authentic products.

[0134] Fig.13 Shown is the characterization of SoGH1. Fig.13(A) Shown is the production of 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1→3)-β-D-pyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRXX, (11)). The structure of [β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1→3)-β-D-pyranosyl-(1→4)-α-L-pyranosyl-rhamnosyl-(1→2)-[β-D-pyranosyl-quinol-(1→4)]-β-D-pyranofucosyl ester}-saponic acid (QA-TriF(Q)RXX, (12)), wherein the background color marked part shows the modification effect of SoGH1; the calculated values ​​of the relevant adducts and fragments of (12) are listed in the attached table. Fig.13 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=1657.7115, i.e., [MH] of (12) - Calculated value of the mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984, SoCSL, SoUGT73DL1, SoUGT73CC6, SoUGT74CD1, SoUGT79T1, SoUGT79L3 and SoUGT73M2) required for the production of QA-TriFRXX (11); under the additional action of SoGH1 activity, a peak corresponding to (12) was generated, which was determined by comparison with the standard authentic product.

[0135] Fig.14 Shown is the characterization of SoBHAD1. Fig.14(A) shows the structure of saponin B (13) generated by SoBAHD1 in combination with saponinase 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-pyranoglucuronic acid-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinolpyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin (QA-TriF(Q)RXX, (12)), wherein the background color marked part shows the modification effect of SoBAHD1; the calculated values ​​of the relevant adducts and fragments of (13) are listed in the attached table. Fig.14 (B) shows representative (n=6) extracted ion chromatograms (EIC) and MS / MS spectra of N. benthamiana leaves transiently co-expressing various genes after extraction and HPLC / MS analysis, wherein the EIC shown is m / z=1699.7206, i.e., [MH] of (13) - Calculated value of the mass of the adduct; the negative control used was an extract of Nicotiana benthamiana leaves co-expressing only AstHMGR (tHMGR control) or co-expressing the soapwort genes (tHMGR, SobAS1, SoCYP716A379, SoCYP72A984, SoCSL, SoUGT73DL1, SoUGT73CC6, SoUGT74CD1, SoUGT79T1, SoUGT79L3, SoUGT73M2 and SoGH1) required for the production of QA-TriF(Q)RXX(12); under the additional action of SoBAHD1 activity, a peak corresponding to (13) was generated, which was determined by comparison with the standard authentic product.

[0136] Fig.15 Shown is the biosynthesis of saponin B. Fig.15 (A) is a bar graph showing the relative accumulation of (4-13) gradually accumulated in Nicotiana benthamiana with the step-by-step expression of each enzyme in the pathway, wherein the relative accumulation was determined by integrating the peak area of ​​the extracted ion current chromatogram; the average value and the error bar representing the average value are plotted in the figure (n=6). Fig.15 (B) Shown is the biosynthetic pathway proposed in this paper for converting epoxysqualene to saponin B (the precursor of saponin A and an isomer of SO1861), in which the structures confirmed by comparison with the authentic standard are marked with black dots; the order of the steps drawn is only a hypothetical sequence proposed, and the actual order in plants may be different.

[0137] Fig.16The figure shows that only limited amounts of β-amyrin (1) were detected in DsRED control hairy roots and SobAS1-silenced hairy roots, where the samples were extracted and analyzed by GC / MS; the figure shows the extracted ion current chromatogram (EIC) at m / z=218; the comparison object was a commercially available β-amyrin standard.

[0138] Fig.17 Shown is a comparison of the GC / MS analysis results of the hairy root sample of Sophora japonica and the cycloartenol standard, wherein the figure shows the extracted ion current chromatogram (EIC) at m / z=408; the peak detected in the mass spectrum is cycloartenol.

[0139] Fig.18 Shown is a comparison of the liquid chromatography-mass spectrometry (LC / MS) analysis results of the hairy root sample of Sophora japonica and the saponin (4) standard, wherein the figure shows the extracted ion current chromatogram (EIC) at m / z = 485.3267 and the fragment peaks detected in the MS / MS analysis.

[0140] Fig.19 Shown is a comparison of the LC / MS analysis results of the Sophora japonica hairy root sample and the saponin B (13) standard, wherein the figure shows the extracted ion current chromatogram (EIC) at m / z = 1699.7206 and the fragment peaks detected in the MS / MS analysis. DETAILED DESCRIPTION

[0141] The present invention relates to the production of triterpenes such as saponins and their intermediates using biosynthetic enzymes encoded by genes and variants newly identified or recognized from the plant Soapwort (Saponaria officinalis). Such enzymes may include: beta-amyrin synthase (bAS; SobAS; SEQ ID NO: 8); SoC28 oxidase (SoC28; SEQ ID NO: 2); SoC23 oxidase (SoC23; SEQ ID NO: 4); C28C16 oxidase (SoC28C16; SEQ ID NO: 6); sapote 3-O glucuronyltransferase (SoQA-GlcAT; SoCSL; SEQ ID NO: 10); QA-GlcA galactosyltransferase (SoC3Gal; SoC3Gal; SEQ ID NO: 12); QA-GlcA-Gal xylosyltransferase (SoQA-R XylT; SoC3Xyl; SEQ ID NO: 14); QA-Tri fucosyltransferase (QATriFuT; SoC28F; SEQ ID NO: 16); QA-TriF rhamnosyltransferase (QA-TriFR; SoC28Rha; SEQ ID NO: 17); ID NO: 18); QA-TriFR xylosyltransferase (SoQA-TriFRXylT; SoC28Xyl1; SEQ ID NO: 20); QA-TriFRX xylosyltransferase (SoQA-TriFRXXylT; SoC28Xyl2; SEQ ID NO: 22); QA-TriFRXX quinotransferase (SoGH1; SEQ ID NO: 34); and / or QA-TriF(Q)RXX acetyltransferase (SoBAHD1; SEQ ID NO: 36).

[0142] Optionally, each gene, polypeptide sequence and nucleotide sequence described herein is obtained or derived from Saponaria officinalis.

[0143] The genes, polypeptide sequences and nucleotide sequences described herein can be used to produce cyclic triterpenes, such as β-amyrin, oleanolic acid, echinocystic acid, saccharic acid (QA), glycosylated forms of saccharic acid (such as saponin, QS-7, QS-21), and analogs and intermediates of such glycosylated forms of saccharic acid.

[0144] In some embodiments, one, two, three, four or more genes described herein can be used to produce saponin (QA). Saponin is a derivative of a simple triterpene, β-amyrin, which is synthesized by cyclizing 2,3-epoxysqualene (OS), a universal linear precursor, with epoxysqualene cyclase (OSC). Further, the C-16, C-23 and C-28 positions of the β-amyrin skeleton are oxidized with alcohols, aldehydes and carboxylic acids, respectively, to form saponin. The linear biosynthetic pathway proposed herein is as follows Fig.15 However, the three oxidation reactions can be carried out in different orders via the corresponding intermediates and achieve the same effect.

[0145] In a preferred embodiment, genes encoding biosynthetic enzymes may be used to produce saponic acid from 2,3-epoxysqualene, as described below.

[0146] 2,3-Epoxysqualene (OS) can be converted into β-amyrin by Sopapilla β-amyrin synthase (SobAS). SobAS may have the amino acid sequence of SEQ ID NO: 8, or a variant or fragment thereof.

[0147] As an alternative, 2,3-epoxysqualene (OS) can also be converted to β-amyrin by endogenous enzymes in the host cell.

[0148] The C-28 position of β-amyrin can be oxidized to a carboxylic acid to generate oleanolic acid by SoC28 oxidase (SoC28). SoC28 oxidase may have an amino acid sequence of SEQ ID NO: 2, or a variant or fragment thereof.

[0149] Subsequently, the C-16 position of oleanolic acid can be oxidized to an alcohol using a saponaria officinalis C28C16 oxidase (SoC28C16) to generate echinocystic acid. The C28C16 oxidase can have the amino acid sequence of SEQ ID NO: 4, or a variant or fragment thereof.

[0150] Alternatively, the C-28 position of β-amyrin can be oxidized to a carboxylic acid and the C-16 position to an alcohol using Soapwort C28C16 oxidase (SoC28C16) to generate echinocystic acid. The SoC28C16 oxidase may have the amino acid sequence of SEQ ID NO: 4, or a variant or fragment thereof.

[0151] The C-23 position of echinocystic acid can be oxidized to an aldehyde to generate quisac acid by SoC23 oxidase (SoC23). SoC23 oxidase can have an amino acid sequence of SEQ ID NO: 6, or a variant or fragment thereof.

[0152] In some embodiments, the genes described herein can be used to glycosylate the C-3 position of saponin. Glycosylation can start from a β-D-glucuronic acid (GlcA) residue attached to the 3-O position of saponin. Subsequently, the GlcA residue is bonded to D-galactose (Gal) via a β-1→2 bond and to D-xylose (Xyl) via a β-1,3 bond.

[0153] In a preferred embodiment, as described below, saponic acid or a C28-glycosylated form of saponic acid can be glycosylated at the 3-O position using a gene encoding a biosynthetic enzyme.

[0154] By Soapwort saponin 3-O glucuronyltransferase (SoQA-GlcAT; SoCSL), D-glucuronic acid (GlcA) can be transferred to the 3-O position of saponin to form 3-O-{β-D-pyranoglucuronic acid}-saponin (QA-GlcA or QA-Mono). SoCSL may have the amino acid sequence of SEQ ID NO: 10, or a variant or fragment thereof.

[0155] By the saponin QA-GlcA galactosyltransferase (SoQA-GalT or SoC3Gal), D-galactose (Gal) can be transferred to QA-Mono (QA-GlcA) through a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal or QA-Di). SoC3Gal may have the amino acid sequence of SEQ ID NO: 12, or a variant or fragment thereof.

[0156] By the saponin QA-GlcA-Gal xylosyltransferase (QA-XylT or SoC3Xyl), D-xylose (Xyl) can be transferred to QA-Di through a 1→3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-[Gal]-Xyl or QA-Tri). QA-XylT may have the amino acid sequence of SEQ ID NO: 14, or a variant or fragment thereof.

[0157] In some embodiments, the genes described herein can be used to glycosylate quince at the C-28 position or the C-3 glycosylated form of quince.

[0158] By the Soapwort QA-Tri fucosyltransferase (SoQA-TriFuT or SoC28Fu), D-fucose (Fuc) can be transferred to the 28-O position of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-soapwort acid (QA-TriF). SoC28Fu may have the amino acid sequence of SEQ ID NO: 16, or a variant or fragment thereof.

[0159] L-rhamnose (Rhap) can be transferred to QA-TriF via a β-1→2 bond by Soapwort QA-TriF rhamnosyltransferase (SoQA-TriFRhaT or SoC28Rha) to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1→2)-β-D-fucopyranosyl ester}-soapwort (QA-TriFR). SoC28Rha may have the amino acid sequence of SEQ ID NO: 18, or a variant or fragment thereof.

[0160] By means of Soapwort QA-TriFR xylosyltransferase (SoQA-TriFRXylT or SoC28Xyl1), D-xylose (Xyl) can be transferred to QA-TriFR via a 1→4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-soapwort acid (QA-TriFRX). SoC28Xyl1 may have the amino acid sequence of SEQ ID NO: 20, or a variant or fragment thereof.

[0161] By means of the soapwort QA-TriFRX xylosyltransferase (SoQA-TriFRXXylT or SoC28Xyl2), D-xylose (Xyl) can be transferred to QA-TriFRX through a 1→3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRXX). SoQA-TriFRXXylT may have the amino acid sequence of SEQ ID NO: 22, or a variant or fragment thereof.

[0162] The quinovosyl group of QA-TriF(Q)RXX can be acetylated by a Soapwort QA-TriF(Q)RXX acetyltransferase (SoBAHD1) polypeptide to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-2)-[β-D-4-O-pyranacetylquinovosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin (SpB). SoBAHD1 may have the amino acid sequence of SEQ ID NO: 36, or a variant or fragment thereof.

[0163] In a preferred embodiment, the methods described herein comprise the use of one or more such newly characterized triterpene biosynthetic nucleic acids (e.g., one, two, three or more such nucleic acids), optionally in combination with the manipulation of other genes known in the art to have an effect on the biosynthesis of saponic acid or glycosylated saponic acid.

[0164] Newly characterized triterpene biosynthetic amino acid and nucleotide sequences from Soapwort (SEQ ID NOs: 1-22 and 33-36), variants of such sequences, and methods of use thereof, themselves constitute various aspects of the present invention. As described in the present disclosure, any such sequence or variant can be used to alter the saponic acid or glycosylated saponic acid content of a plant. For example, a variant nucleic acid may include a sequence encoding a variant polypeptide having the relevant biological activity of a native polypeptide as described above. Such variants include, for example, variants of any of SEQ ID NOs: 1-22 and 33-36. For the sake of brevity, in the context of the present invention, particularly in the context of the methods and uses described herein, the polypeptides or nucleotide sequences of SEQ ID NOs: 1-22 and 33-36 described herein and their variants, such as triterpene biosynthetic genes and triterpene biosynthetic polypeptides, may be referred to herein as "triterpene biosynthetic sequences".

[0165] The present application provides a SobAS polypeptide, which has an amino acid sequence of SEQ ID NO: 8 or a variant thereof, such as an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8. The present application also provides: a nucleic acid encoding the SobAS polypeptide, which has a nucleotide sequence of SEQ ID NO: 7 or a variant thereof, such as a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7; and a vector comprising the nucleic acid. The SobAS polypeptide may have the function of cyclizing the universal linear precursor 2,3-epoxysqualene (OS) to triterpenes.

[0166] The present application also provides a SoC28 oxidase polypeptide, which has an amino acid sequence of SEQ ID NO: 2 or a variant thereof, such as an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2. The present application also provides: a nucleic acid encoding the SoC28 oxidase polypeptide, which has a nucleotide sequence of SEQ ID NO: 1 or a variant thereof, such as a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 1; and a vector comprising the nucleic acid. The SoC28 oxidase polypeptide may have the function of oxidizing the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid.

[0167] The present application also provides a SoC28C16 oxidase polypeptide, which has an amino acid sequence of SEQ ID NO: 4 or a variant thereof, such as an amino acid sequence with at least 50% sequence identity to SEQ ID NO: 4. The present application also provides: a nucleic acid encoding the SoC28C16 oxidase polypeptide, which has a nucleotide sequence of SEQ ID NO: 3 or a variant thereof, such as a nucleotide sequence with at least 50% sequence identity to SEQ ID NO: 3; and a vector comprising the nucleic acid. The SoC28C16 oxidase polypeptide may have the function of oxidizing the C-16 position of β-amyrin to alcohol and oxidizing the C-28 position thereof to carboxylic acid to form echinocystic acid.

[0168] The present application also provides a SoC23 oxidase polypeptide, which has an amino acid sequence of SEQ ID NO: 6 or a variant thereof, such as an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 6. The present application also provides: a nucleic acid encoding the SoC23 oxidase polypeptide, which has a nucleotide sequence of SEQ ID NO: 5 or a variant thereof, such as a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 5; and a vector comprising the nucleic acid. The SoC23 oxidase may have the function of oxidizing the C-23 position of echinocystic acid to an aldehyde to form sapoteic acid.

[0169] The present application also provides a soapwort saponin 3-O glucuronyl transferase (SoCSL) polypeptide, which has an amino acid sequence of SEQ ID NO: 10 or a variant thereof, such as an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 10. The present application also provides: a nucleic acid encoding the saponin 3-O glucuronyl transferase (SoCSL) polypeptide, which has a nucleotide sequence of SEQ ID NO: 9 or a variant thereof, such as a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: 9; and a vector comprising the nucleic acid. SoCSL may have the function of attaching D-glucuronic acid (GlcA) to the 3-O position of saponin to form 3-O-{β-D-pyranoglucuronic acid}-saponin (QA-GlcA or QA-Mono).

[0170] The present application also provides a soapwort QA-GlcA galactosyltransferase (SoC3Gal) polypeptide, which has an amino acid sequence of SEQ ID NO: 12 or a variant thereof, such as an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 12. The present application also provides: a nucleic acid encoding the soapwort QA-GlcA galactosyltransferase (SoC3Gal) polypeptide, which has a nucleotide sequence of SEQ ID NO: 11 or a variant thereof, such as a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 11; and a vector comprising the nucleic acid. SoC3Gal may have the function of attaching D-galactose (Gal) to QA-GlcA through a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-Di or QA-GlcA-Gal).

[0171] The present application also provides a soapwort QA-GlcA-Gal xylose transferase (SoC3Xyl) polypeptide, which has an amino acid sequence of SEQ ID NO: 14 or a variant thereof, such as an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 14. The present application also provides: a nucleic acid encoding the soapwort QA-GlcA-Gal xylose transferase (SoC3Xyl) polypeptide, which has a nucleotide sequence of SEQ ID NO: 13 or a variant thereof, such as a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 13; and a vector comprising the nucleic acid. SoC3Xyl may have the function of attaching D-xylose (Xyl) to QA-GlcA-Gal through a 1,3 bond to form 1,3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcAGalXyl or QA-Tri).

[0172] The present application also provides a Saponaria officinalis QA-Tri fucosyltransferase (SoC28Fu) polypeptide, which has an amino acid sequence of SEQ ID NO: 16 or a variant thereof, such as an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 16. The present application also provides: a nucleic acid encoding the Saponaria officinalis QA-Tri fucosyltransferase (SoC28Fu) polypeptide, which has a nucleotide sequence of SEQ ID NO: 15 or a variant thereof, such as a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: 15; and a vector comprising the nucleic acid. SoC28Fu may have the function of attaching fucose (Fuc) to the 28-O position of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponic acid (QA-TriF).

[0173] The present application also provides a Saponaria officinalis QA-TriF rhamnosyltransferase (SoC28Rha) polypeptide, which has an amino acid sequence of SEQ ID NO: 18 or a variant thereof, such as an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 18. The present application also provides: a nucleic acid encoding the Saponaria officinalis QA-TriF rhamnosyltransferase (SoC28Rha) polypeptide, which has a nucleotide sequence of SEQ ID NO: 17 or a variant thereof, such as a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 17; and a vector comprising the nucleic acid. SoC28Rha may have the function of attaching rhamnose (Rha) to QA-TriF through a 1,2 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-pyranofucoid ester}-saponic acid (QA-TriFR).

[0174] The present application also provides a Soapwort QA-TriFR xylose transferase (SoC28Xyl1) polypeptide, which has an amino acid sequence of SEQ ID NO: 20 or a variant thereof, such as an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 20. The present application also provides: a nucleic acid encoding the Soapwort QA-TriFR xylose transferase (SoC28Xyl1) polypeptide, which has a nucleotide sequence of SEQ ID NO: 19 or a variant thereof, such as a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 19; and a vector comprising the nucleic acid. SoC28Xyl1 can have the function of attaching D-xylose (Xyl) to QA-TriFR through a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponic acid (QA-TriFRX).

[0175] The present application also provides a Soapwort QA-TriFRX xylose transferase (SoC28Xyl2) polypeptide, which has an amino acid sequence of SEQ ID NO: 22 or a variant thereof, such as an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 22. The present application also provides: a nucleic acid encoding the Soapwort QA-TriFRX xylose transferase (SoC28Xyl2) polypeptide, which has a nucleotide sequence of SEQ ID NO: 21 or a variant thereof, such as a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 21; and a vector comprising the nucleic acid. SoC28Xyl2 can have the function of attaching D-xylose (Xyl) to QA-TriFRX through a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponic acid (QA-TriFRXX).

[0176] The present application also provides a Soapwort QA-TriFRXX quinoline transferase (SoGH1) polypeptide, which has an amino acid sequence of SEQ ID NO: 34 or a variant thereof, such as an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 34. The present application also provides: a nucleic acid encoding the Soapwort QA-TriFRXX quinoline transferase (SoGH1) polypeptide, which has a nucleotide sequence of SEQ ID NO: 33 or a variant thereof, such as a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 33; and a vector comprising the nucleic acid. SoGH1 may have the function of attaching D-quinovose (Q) to QA-TriFRXX through a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponic acid (QA-TriF(Q)RXX).

[0177] The present application also provides a Soapgrass QA-TriF(Q)RXX acetyltransferase (SoBAHD1) polypeptide, which has an amino acid sequence of SEQ ID NO: 36 or a variant thereof, such as an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 36. The present application also provides: a nucleic acid encoding the Soapgrass QA-TriF(Q)RXX acetyltransferase (SoBAHD1) polypeptide, which has a nucleotide sequence of SEQ ID NO: 35 or a variant thereof, such as a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 35; and a vector comprising the nucleic acid. SoBAHD1 may have the function of forming 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-4-O-pyranacetylquinovosyl-(1→4)]-β-D-fucopyranosyl ester}-saponic acid (SpB) by acetylation of QA-TriF(Q)RXX.

[0178] An amino acid sequence described herein is a variant of a reference sequence, and one or more amino acid residues may be altered relative to the reference sequence, such as a peptide, polypeptide or protein sequence described herein, and for example any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 34 or 36. For example, 50 or less, preferably 45 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 5 or less, or 3 or less, 2 or 1 amino acid residues may be altered relative to the reference sequence. For example, a variant described herein may include a sequence having 50 or less, 45 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 5 or less, 3 or less, 2 or 1 mutant amino acid residues based on the reference sequence.

[0179] The amino acid residues in the reference sequence can be changed or mutated by insertion, deletion or substitution, preferably replaced with different amino acid residues. Such changes can be caused by one or more of the addition, insertion, deletion or substitution of one or more nucleotides in the encoding nucleic acid.

[0180] A nucleotide sequence described herein is a variant of a reference sequence, and one or more nucleotides may be changed relative to the reference sequence, such as a nucleotide sequence described herein, and for example, any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 33 or 35. For example, 50 or less, preferably 45 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 5 or less, or 3 or less, 2 or 1 nucleotides may be changed relative to the reference sequence. For example, a variant described herein may include a sequence having 50 or less, 45 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 5 or less, 3 or less, 2 or 1 mutant nucleotides based on the reference sequence.

[0181] A peptide, polypeptide or protein described herein or a nucleotide sequence described herein is a variant of a reference sequence such as the above amino acid or nucleotide sequence, and may have at least 50% sequence identity with the reference sequence, the sequence identity is at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98% or at least 99%. For example, a protein variant described herein may include an amino acid sequence having at least 50% sequence identity with a reference amino acid sequence, a sequence identity of at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98% or at least 99% with the reference amino acid sequence, such as one or more of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 34 and 36.

[0182] A nucleic acid variant described herein may include a nucleotide sequence having at least 50% sequence identity to a reference amino acid sequence, at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to a reference nucleotide sequence, such as one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 33 and 35.

[0183] Different variant triterpene biosynthetic sequence variants may have different degrees of sequence identity with their corresponding reference sequences. Various combinations of variant triterpene biosynthetic sequences having all of the above sequence identity degrees are encompassed by the present invention.

[0184] Sequence identity is usually determined in conjunction with the GAP algorithm (Wisconsin GCG software package, Accelerys Inc, San Diego, USA). The GAP algorithm uses the Needleman-Wunsch algorithm to align two complete sequences in a manner that maximizes the number of matches and minimizes the number of gaps. In general, the following default parameters are used: gap creation penalty = 12; gap extension penalty = 4. Although the GAP algorithm is preferably used, other algorithms may also be used, such as the BLAST algorithm (using the method of Altschul et al., J. Mol. Biol., 1990, 215, 405-410), the FASTA algorithm (using the method of Pearson and Lipman, PNAS USA, 1988, 85, 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman, J. Mol. Biol., 1981, 147, 195-197), or the TBLASTN program of Altschul et al. (1990, supra), wherein the program generally uses default parameters. Among them, the site-specific iterative basic local alignment search tool (PSI-BLAST) algorithm (Nucl. Acids Res., 1997, Vol. 25, pp. 3389-3402) can be used in particular. In addition, the GenomeQuest TM Sequence identity and similarity were determined using the Gene-IT software (Gene-IT, Worcester, MA, USA).

[0185] Preferably, the sequence alignment of related sequences described herein is a full-length alignment.

[0186] Variant polypeptides may have the relevant biological activity of the reference polypeptide. Variant nucleic acids may encode relevant variant polypeptides. In the context of the present invention, the "biological activity" of a polypeptide as described herein refers to the catalytic activity of Fig.15 The relevant biological activity can be determined according to Fig.15 Alternatively, the relevant biological activity can also be analyzed according to the in vivo activity in the manner described in the examples. That is, by introducing a variety of heterologous constructs, the corresponding products are generated in the host, and then the products can be analyzed by LC / MS and the like.

[0187] Preferred variants may be:

[0188] (i) Natural nucleic acids, such as alleles (including polymorphisms or mutations of one or more bases) or pseudo-alleles (which may exist at a locus closely linked to the biosynthetic genes described herein). In addition, paralogous genes, isogenes or other homologous genes belonging to the same family (such as belonging to the same evolutionary branch or sub-evolutionary branch) as the biosynthetic genes described herein are also included. In addition, orthologous genes or homologous genes from other plant species (i.e., plants other than soapwort) are also included. As described below, homology can be at the nucleotide sequence level and / or the amino acid sequence level.

[0189] (ii) Artificial nucleic acids that can be prepared by one skilled in the art based on the present disclosure. Such derivatives can be prepared, for example, by site-directed mutagenesis, random mutagenesis, or direct synthesis. Variant nucleic acids are preferably generated directly or indirectly (e.g., by one or more amplification or replication steps) from an original nucleic acid having the entire or partial sequence of a biosynthetic gene described herein.

[0190] Variants may also include nucleic acids corresponding to the above nucleic acids but extended at the 3' end or the 5' end.

[0191] A method of generating a variant triterpene biosynthetic nucleic acid may comprise the steps of modifying any of the genes described herein, such as one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 33 and 35.

[0192] For a variety of reasons, it may be necessary to modify. For example, by modification, restriction endonuclease sites can be introduced or removed, or the use of codons can be changed. When it is necessary to express the above-mentioned gene in an alternative host (such as a microbial host such as yeast), such modification may be particularly necessary. For this purpose, there are known codon optimization gene methods in the art (for example, see Elena, Claudia et al., "Expression of codon optimized genes in microbial systems: current industrial applications and viewpoints", Frontiers in microbiology, 2014, 5 issues). The sequences described herein containing codon modification points for maximizing yeast expression constitute embodiments of the present invention.

[0193] As an alternative, the sequence can also be modified to generate a derivative by adding, inserting, deleting or replacing one or more (such as several) of one or more nucleotides in the nucleic acid, thereby achieving the addition, insertion, deletion or replacement of one or more (such as several) amino acids in the encoded polypeptide.

[0194] Such modifications may alter: sites required for post-translational modification, such as cleavage sites in the encoded polypeptide; phosphorylation motifs in the encoded polypeptide. When it is necessary to isolate the expressed protein from a microbial system, a leader or other guide sequence (such as a membrane localization sequence or a Golgi localization sequence) may be added to it to determine its location after expression.

[0195] In addition, random mutation or site-directed mutation may be required to change the activity (such as specificity) or stability of the encoded polypeptide. Among them, such changes can be achieved by conservative variation, that is, replacing one hydrophobic residue such as isoleucine, valine, leucine or methionine with another residue, or replacing one polar residue with another polar residue, for example, replacing lysine with arginine, replacing aspartic acid with glutamic acid, or replacing asparagine with glutamine. As a well-known fact to those skilled in the art, changing the primary structure of a polypeptide by conservative substitution may not lead to significant changes in the activity of the polypeptide. The reason is that the side chain of the amino acid in the inserted sequence can form similar bonds and contact conditions with the side chain of the replaced amino acid. Among them, this is true even when the substitution occurs in a region that plays a key role in determining the conformation of the peptide. In addition, variants containing non-conservative substitution points are also included. As a well-known fact to those skilled in the art, when the substitution occurs in a region that does not play a key role in determining the conformation of the peptide, the three-dimensional structure of the peptide may not be greatly changed and its activity will not be greatly affected. When such variations occur in regions that play a key role in determining polypeptide conformation or activity, they may result in favorable properties for the polypeptide. In fact, such variations may result in slightly favorable properties for the peptide, for example, changing its stability or specificity.

[0196] In some embodiments, a variant nucleotide sequence encoding such a polypeptide can be obtained by a method comprising the steps of:

[0197] (a) Providing a nucleic acid preparation, such as a nucleic acid preparation derived from a plant cell. The nucleic acid to be tested can be provided by the cell as genomic DNA, cDNA or RNA, or as a mixture of any such substances, preferably as a library in a suitable vector. When genomic DNA is used, a probe can be used to identify a non-transcribed region of the gene (such as a promoter, etc.), such as the region described below;

[0198] (b) providing a nucleic acid molecule as a probe or primer as described above;

[0199] (c) contacting the nucleic acid in the preparation with the nucleic acid molecule under conditions such that the nucleic acid molecule hybridizes with any of the genes or homologous genes in the preparation; and

[0200] (d) Identifying a gene or homologous gene by hybridization with a nucleic acid molecule when the gene or homologous gene is present. The binding of the probe to the target nucleic acid (e.g., DNA) may be measured by any of a variety of techniques at the discretion of one skilled in the art. For example, the probe may be labeled radioactively, fluorescently, or enzymatically. Other methods that do not employ probe labeling include: PCR amplification (see below); ribonuclease cleavage; and allele-specific oligonucleotide probe detection. After successful hybridization is determined, the hybridized nucleic acids are separated. The separation may involve one or more steps of PCR or amplification of the vector in a suitable host.

[0201] Among them, preliminary experiments can be performed by hybridization under less stringent conditions. For probe detection methods, conditions with a stringency sufficient to obtain a simple pattern with a small number of hybridizations identified as positive for subsequent investigation are preferred.

[0202] Hybridization can be performed, for example, according to the method of Sambrook et al. (see below), which uses a hybridization solution containing the following components: 5×SSC (wherein SSC=0.15M sodium chloride; 0.15M sodium citrate; pH=7); 5×Denhardt reagent; 0.5-1.0% SDS; 100 μg / ml denatured and fragmented salmon sperm DNA; 0.05% sodium pyrophosphate; and less than 50% formamide. Hybridization is performed at 37-42°C for at least six hours. After hybridization, the filter is washed as follows: (1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1% SDS; (3) 30 minutes to 1 hour at 37°C in 1×SSC and 1% SDS; (4) 2 hours at 42-65°C in 1×SSC and 1% SDS; the solution is replaced every 30 minutes.

[0203] A commonly used formula for calculating the stringency of hybridization between nucleic acid molecules with specific sequence homology (Sabrook et al., 1989) is: m =81.5°C+16.6Log[Na+]+0.41(%G+C)-0.63(%formamide)-600 / number of duplex base pairs.

[0204] To illustrate the above calculation, assume that [Na+] = [0.368], formamide = 50%, GC content = 42% and average probe size = 200 bases, then T m =57°C. For every 1% increase in homology, the T of the DNA duplex increases. m Reduce by 1 to 1.5° C. Therefore, at a hybridization temperature of 42° C., when the sequence identity of the target nucleic acid is greater than about 75%, it can be observed. Such sequences are considered to be substantially homologous to the nucleic acid sequence of the present invention.

[0205] A well-known practice in the art is to gradually increase the stringency of hybridization until only a few positive clones remain. Other suitable conditions include, for example, hybridization overnight at 42°C in 0.25M Na2HPO4 (pH=7.2, 6.5% SDS, 10% dextran sulfate) when detecting sequences with an identity of about 80-90%, and finally washing with 0.1×SSC (0.1% SDS) at 55°C. When detecting sequences with an identity greater than about 90%, suitable conditions include hybridization overnight at 65°C in 0.25M Na2HPO4 (pH=7.2, 6.5% SDS, 10% dextran sulfate), and finally washing with 0.1×SSC (0.1% SDS) at 60°C.

[0206] In another embodiment, the hybridization of triterpene biosynthetic nucleic acid molecules and variants can be determined or confirmed indirectly by nucleic acid amplification reaction, especially polymerase chain reaction (PCR). Since PCR needs to use two kinds of primer specific amplification target nucleic acids, it is preferred to use two kinds of nucleic acid molecules of sequence characterization triterpene biosynthetic genes. When using cDNA end rapid amplification (RACE) PCR, only one such primer can be needed (see "PCR operation guide collection: method and application guide" (PCR protocols; A Guide to Methods and Applications), Innis (Innis) et al., Academic Press (Academic Press), New York, 1990).

[0207] Thus, a method described herein involving the use of PCR to obtain a variant triterpene biosynthetic nucleic acid may comprise:

[0208] (a) providing a plant nucleic acid preparation, the plant nucleic acid being derived, for example, from seeds or other suitable tissues or organs;

[0209] (b) providing a pair of nucleic acid molecule primers that can be used for (i.e., suitable for) PCR, wherein at least one of the two primers is a primer for the triterpene biosynthesis sequence as described above;

[0210] (c) contacting the nucleic acid in the preparation with primers under conditions for performing PCR;

[0211] (d) performing PCR and determining whether an amplified PCR product is present. If an amplified PCR product is present, it may indicate that a variant has been identified.

[0212] In all of the above cases, clones or fragments identified during the search can be expanded as needed. For example, if it is suspected to be incomplete, the original DNA source (such as a clone library, mRNA preparation, etc.) can be returned to isolate the missing portion, for example by sequence, probe or primer, and based on the portion obtained to identify other clones containing overlapping sequences.

[0213] The methods described herein can use fragments of the triterpene biosynthetic genes described herein (such as one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 33 and 35), or fragments of variants of such genes. In addition, fragments of the full-length polypeptides disclosed herein, especially the production and use of active portions thereof, are also provided. An "active portion" of a polypeptide refers to a peptide that is smaller than the full-length polypeptide but retains its basic biological activity (e.g., biological activity associated with the production of sapogenin or the glycosylation of sapogenin).

[0214] A fragment of a full-length reference triterpene biosynthetic polypeptide sequence such as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 34 or 36 is a contiguous amino acid sequence derived from the full-length protein sequence, which amino acid sequence consists of at least one less amino acid than the full-length protein sequence. For example, compared to the full-length sequence, the fragment may lack a sequence of 10 or more, 20 or more, 50 or more, or 100 or more amino acids. Preferably, the fragment has the relevant biological activity of the full-length reference polypeptide.

[0215] In some embodiments, a polypeptide fragment may include one or more epitopes that can be used to generate antibodies against a portion of any amino acid sequence of the present disclosure. Preferably, the epitope is an epitope that can be specifically bound by an antibody, wherein specific binding can be considered to have a binding affinity to a polypeptide or fragment thereof that is at least about 1000 times greater than the binding affinity to other polypeptides.

[0216] Purified proteins (polypeptides, enzymes) or fragments, mutants, derivatives or variants thereof (eg produced recombinantly by expression of corresponding nucleic acids encoding triterpene biosynthesis) form an aspect of the present invention.

[0217] Such purified polypeptides can be used to generate antibodies by standard techniques in the art. Antibodies and polypeptides comprising antigen-binding fragments of antibodies can be used to recognize homologous genes from species, as further described below.

[0218] The method for producing an antibody comprises: immunizing a mammal (such as a human, mouse, rat, rabbit, horse, goat, sheep or monkey) with a protein or a fragment thereof. After the animal is immunized, antibodies can be obtained from the animal by any of the various techniques known in the art, and the antibodies can be screened, preferably by binding of the antibodies to the target antigen. Among them, for example, protein immunoblotting or immunoprecipitation techniques can be used (Armitage et al., 1992, Nature, Vol. 357, pp. 80-82). Antibodies can be either polyclonal antibodies or monoclonal antibodies.

[0219] As an alternative or in addition to immunizing a mammal, antibodies with appropriate binding specificities may be obtained from a library of recombinantly produced immunoglobulin variable domains expressed, for example, from lambda phage or filamentous phage having functional immunoglobulin binding domains on their surface (see, e.g., WO92 / 01047).

[0220] Antibodies raised against polypeptides or peptides can be used to identify and / or isolate homologous polypeptides and, in turn, their encoding genes.

[0221] Antibodies can be modified in a variety of ways. Indeed, the term "antibody" should be understood to encompass any specific binding substance having a binding domain with the desired specificity. Thus, the term encompasses antibody fragments as well as derivatives, functional equivalents and homologs of antibodies, including any polypeptide containing a natural or synthetic immunoglobulin binding domain.

[0222] Mevalonic acid (MVA) is an important intermediate in the synthesis of triterpenes. Therefore, in order to achieve the highest production of triterpenes such as saponic acid, it may be necessary to express rate-limiting MVA pathway genes in the host.

[0223] HMG-CoA reductase (HMGR) is considered to be a rate-limiting enzyme in the MVA pathway.

[0224] It has been demonstrated that the triterpene (β-amyrin) content can be increased by transiently expressing a recombinant non-feedback-sensitive truncated form of HMGR (tHMGR) in Nicotiana benthamiana (Reed J et al., Metab Eng, 2017, 42, 185-193).

[0225] In some embodiments, a heterologous HMGR (eg, a non-feedback sensitive HMGR) may be used with the triterpene biosynthesis genes described herein.

[0226] The HMGR coding sequence or polypeptide sequence includes, for example, SEQ ID NOs: 23 to 26, or variants or fragments thereof. The variant may be a homologous gene, allele, or artificial derivative as described above in conjunction with the above-mentioned biosynthetic gene or polypeptide. For example, the natural HMGR of the host, such as yeast HMGR in a yeast host, may be preferably used. HMGR genes are known in the art and may be selected as appropriate according to the present disclosure.

[0227] In addition, squalene synthase (SQS) has been reported to be a potential rate-limiting step (Reed et al., supra).

[0228] In some embodiments, a heterologous SQS can be used with the biosynthetic genes described herein, and optionally with the HMGR described herein.

[0229] The SQS coding sequence or polypeptide sequence includes, for example, SEQ ID NO: 27-28, or a variant or fragment thereof. The variant may be a homologous gene, allele, or artificial derivative as described above in conjunction with the above-mentioned biosynthetic gene or polypeptide. For example, the natural SQS of the host, such as yeast SQS in a yeast host, may be preferably used. SQS genes are known in the art and may be selected as appropriate according to the present disclosure.

[0230] When using a specific host (such as yeast), it may be necessary to introduce other genes to increase the flux of biosynthetic production. Such other genes may, for example, include one or more plant cytochrome P450 reductases (CPRs) to serve as reduction partners for the introduced P450. In some embodiments, a heterologous cytochrome P450 reductase such as AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2) may be used in conjunction with the biosynthetic polypeptides and genes described herein. The AtATR2 coding sequence or polypeptide sequence, for example, includes SEQ ID NOs: 29 to 30, or variants or fragments thereof. Variants may be homologous genes, alleles, or artificial derivatives, etc., as described above in conjunction with the above-mentioned biosynthetic polypeptides and genes.

[0231] In some embodiments, a heterologous nucleic acid described herein may further encode one or more of the following polypeptides: (i) HMG-CoA reductase (HMGR) and / or squalene synthase (SQS). Alternatively, HMGR or SQS may be selected from the corresponding polypeptides of SEQ ID NOs: 24, 26 and 28, or variants or fragments of any such polypeptides, or may be encoded by the corresponding polynucleotides of SEQ ID NOs: 23, 25 and 27, or variants or fragments of any such polynucleotides.

[0232] Nucleic acids may include cDNA, RNA, genomic DNA, and modified nucleic acids or nucleic acid analogs (such as peptide nucleic acids). Once a DNA sequence is indicated (for example, indicated in conjunction with a picture), it covers the RNA equivalent of replacing T with U unless the context requires otherwise. Nucleic acids may include more than one nucleic acid molecule. The nucleic acid molecules of the present invention may be provided, separated and / or purified from their natural environment in a substantially pure or homogeneous form, or may not contain or substantially contain other nucleic acids of the source species, and may be double-stranded or single-stranded. In this article, the term "isolated" encompasses all possible forms thereof. Nucleic acid molecules may be synthetic molecules in whole or in part. Nucleic acid molecules may be recombinant nucleic acid molecules in the following sense: nucleic acid sequences that are not connected (not adjacent) in nature are coordinated and connected, or artificially combined in other ways. Nucleic acids may include any of the following sequences, or consist of them, or consist essentially of them.

[0233] The complementary strand of a nucleic acid described herein refers to the complementary sequence of the nucleic acid, or the complementary sequence of the nucleotide sequence contained in the nucleic acid. Optionally, compared with the reference nucleotide sequence, the complementary sequence is the full-length sequence.

[0234] As used herein, the term "heterologous" is a broad term, meaning that the gene / sequence of the target nucleotide (e.g., encoding a biosynthetic modified polypeptide) has been introduced into the cells of the host or its ancestors by genetic modification (i.e., by human intervention). The presence of a nucleic acid heterologous to a host cell in a cell of that type, variety, or species is non-natural. Thus, a heterologous nucleic acid may include a coding sequence of a specific type of plant cell or a plant of a specific species or variety placed in the environment of other types of plant cells or plants of other species or varieties, or a coding sequence derived from the plant cell or plant. In another possibility, the nucleic acid sequence or its homologous gene is naturally present in the cell into which the nucleic acid sequence is to be placed, but the nucleic acid sequence is connected and / or adjacent to a nucleic acid that is non-natural to the cell or the plant cell of that type, species, or variety. For example, it is operably connected to one or more regulatory sequences for controlling expression, such as a promoter sequence.

[0235] In the context of the present invention, "transformation" refers to the nucleotide sequence of a heterologous nucleic acid causing one or more properties of a cell to change, thereby changing its phenotype. For example, the phenotype of its ability to biosynthesize triterpenes such as sapogenin or glycosylated sapogenin (such as QATri, QATriFRXX, QATriF(Q)RXX or SpB) is changed. Such transformation can be either transient or stable.

[0236] "Not capable of biosynthesis" means that the host does not or is not thought to naturally produce detectable or recoverable levels of the product under its normal metabolic conditions prior to transformation. After application of the present invention, the host becomes capable of producing detectable or recoverable levels of the product.

[0237] The nucleotide sequence information provided herein can be used to design probes and primers used for probe detection or amplification. The length of the oligonucleotides used for probe detection or PCR can be about 30 or less (such as 18, 21 or 24) nucleotides. Generally, the length of the specific primer is more than 14 nucleotides. In order to obtain the best specificity and cost-effectiveness, the primer length can preferably be 16 to 24 nucleotides. Those skilled in the art should be proficient in designing primers used in processes such as PCR. As needed, the complete restriction fragments of the disclosed gene can be used for probe detection, and the length of the complete restriction fragments can be hundreds or even thousands of nucleotides. As needed, smaller variations can be introduced to generate "universal" or "degenerate" primers.

[0238] Probe detection can be performed using standard Southern blotting techniques. For example, DNA can be extracted from cells and digested with different restriction endonucleases. After this, the restriction fragments can be separated by agarose gel electrophoresis and then denatured and transferred to a nitrocellulose filter. Thereafter, the labeled probe can be hybridized to the single-stranded DNA fragments on the filter and its binding status can be determined. The DNA used for probe detection can be prepared by RNA preparations derived from cells. Alternatively, probe detection can be performed by so-called "nucleic acid chips" (see the review article by Marshall and Hodgson, 1998, Nature Biotechnology, 16, 27-31).

[0239] One method described herein can be to co-infiltrate multiple strains of Agrobacterium tumefaciens to achieve coordinated expression according to the above biosynthetic pathway, wherein each strain carries one or more triterpene biosynthetic genes as described above.

[0240] In some embodiments, at least two or three different Agrobacterium tumefaciens strains are co-infiltrated, each strain, for example, carrying a triterpene biosynthetic nucleic acid.

[0241] The above genes can be derived from transient expression vectors.

[0242] Vectors (generally binary vectors) used in the manner described herein may generally include an expression cassette comprising:

[0243] (i) a promoter operably linked to:

[0244] (ii) an enhancer sequence derived from the RNA-2 genomic segment of a bipartite RNA virus, wherein the target start site in the RNA-2 genomic segment has been mutated;

[0245] (iii) a nucleic acid sequence encoding one or more biosynthetic genes as described above;

[0246] (iv) a terminator sequence; and optionally,

[0247] (v) a 3' untranslated region (UTR) located upstream of the terminator sequence.

[0248] Further examples of vectors and expression systems suitable for use in the manner described herein are given below.

[0249] The triterpene biosynthesis genes described above may be contained in or in the form of a preferably replicable recombinant vector. The vector may include any plasmid, cosmid, phage or Agrobacterium binary vector in linear or circular double-stranded or single-stranded form, which may or may not be capable of self-propagation or mobility, and which may transform a prokaryotic or eukaryotic host by integration into the cell genome or by presence outside the chromosome (e.g., an autonomously replicating plasmid with a replication origin).

[0250] Suitable expression vectors may include binary vectors for Agrobacterium tumefaciens-mediated transient expression (e.g., see Bevan et al., Nucleic Acids Research, November 26, 1984, Vol. 12, No. 22, pp. 8711-872). It is well known to those skilled in the art that the "binary vector" system includes: (a) a border sequence that allows the desired nucleotide sequence to be transferred into the genome of a plant cell; (b) the desired nucleotide sequence itself, which generally includes an expression frame of the following two operably linked as appropriate: (i) an active promoter of a plant; (ii) a target sequence and / or an enhancer. The desired nucleotide sequence is located between the border sequences and can be inserted into the plant genome under appropriate conditions. The binary vector system generally requires other sequences (derived from Agrobacterium tumefaciens) to achieve integration. Generally, this purpose can be achieved by the so-called "Agrobacterium infiltration method", which uses Agrobacterium-mediated transient transformation. In short, this technology is based on the property that Agrobacterium tumefaciens transfers part of its DNA (T-DNA) to a host cell and can be integrated into the nuclear DNA in the host cell. The T-DNA is defined by a left border sequence and a right border sequence of approximately 21 to 23 nucleotides in length. Infiltration can be achieved, for example, by syringe (for leaves) or vacuum (for whole plants). In the present invention, the border sequences are generally contained near the desired nucleotide sequence (T-DNA), and one or more vectors are introduced into the plant material by means of Agrobacterium infiltration.

[0251] Other suitable expression systems may use the so-called "Hyper-Translatable" cowpea mosaic virus (CPMV-HT) system. Suitable vectors based on the pEAQ-HT expression plasmid used in the CPMV-HT system are well known in the art (see, for example, WO2009 / 087391; Sainsbury et al., Plant Biotechnol J, 2009, Vol. 7, No. 7, pp. 682-693).

[0252] In general, those skilled in the art should have good vector construction and recombinant gene expression scheme (for example, for expressing heterologous nucleic acid in the host or one or more cells of the host) design capabilities. Suitable vectors can be obtained by selection or construction, and include suitable regulatory sequences, which include promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. More details are shown in "Molecular Cloning: a Laboratory Manual, Second Edition, Sabrook et al., 1989, Cold Spring Harbor Laboratory Press" or "Current Protocols in Molecular Biology, Second Edition, Ausubel et al., John Wiley & Sons, 1992).

[0253] Specifically included are shuttle vectors, which are DNA vectors that are naturally or designed to replicate in different host organisms, which host organisms can be selected from actinomycetes and related species, bacteria, and eukaryotic organisms (such as higher plants, mosses, yeast or fungal cells).

[0254] A vector comprising a nucleic acid described herein need not include a promoter or other regulatory sequences, particularly when used for recombination into the genome by introducing the nucleic acid into a cell.

[0255] Preferably, the nucleic acid in the vector is controlled by a suitable promoter or other regulatory elements for transcription into a host cell such as a microbial cell (such as a yeast cell and a bacterial cell) or a plant cell, and is operably connected thereto. The vector can be a bifunctional expression vector that works in a variety of hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory elements (optionally used in combination with heterologous enhancers such as the 35S enhancer described in the embodiments below). The advantage of using a natural promoter is that pleiotropic reactions can be avoided. In the case of cDNA, it may be controlled by a suitable promoter or other regulatory elements for expression in a host cell.

[0256] A promoter is a nucleotide sequence that allows the DNA operably linked downstream of the promoter (ie, in the 3' direction of the sense strand of the double-stranded DNA) to initiate transcription from the promoter.

[0257] "Operably linked to" means connected to become part of the same nucleic acid molecule, and the position and orientation are suitable for initiating transcription from the promoter. DNA operably linked to a promoter is under the "transcription initiation control" of the promoter.

[0258] Suitable promoters include inducible promoters. Those skilled in the art will fully understand the meaning of the term "inducible" before the promoter. In essence, the expression controlled by an inducible promoter is a "switch-activated" expression, or an expression that is enhanced in response to an applied stimulus. The stimulation of different promoters has different properties. In the absence of a suitable stimulus, some inducible promoters will result in a very small amount of expression, or the expression level caused will be so low that it cannot be detected (or will not result in expression). Other inducible promoters will result in detectable constitutive expression in the absence of a stimulus. Regardless of the expression level in the absence of a stimulus, any inducible promoter will enhance expression when a suitable stimulus is present.

[0259] The nucleic acids described herein can be placed under the control of an external inducible gene promoter to place the expression (of the heterologous sequence) under the control of the user. The advantage of introducing a heterologous gene into a plant cell (especially when the cell is contained in a plant) is that the expression of the gene can be placed under the control of a selected promoter, thereby being able to affect gene expression as desired, thereby affecting the biosynthesis of sapogenin or glycosylated sapogenin. In addition, mutants and derivatives of wild-type genes (whose activity, for example, is higher or lower than that of the wild-type) can be used to replace endogenous genes.

[0260] In addition, a gene construct, preferably a replicable vector, is provided, comprising a promoter (optionally an inducible promoter) operably linked to the biosynthetic gene or variant thereof described herein.

[0261] In the context of the present invention, nucleic acid constructs that operate as plant vectors are of particular interest. Various procedures and vectors have been used with widespread success in plants, as described in Guerineau and Mullineaux (1993) (the chapter "Plant Transformation and Expression Vectors" (pages 121-148) in Plant Molecular Biology Labfax, edited by Croy R.R.D. and published by BIOS Scientific Publishers, Oxford). Suitable vectors may include plant virus-derived vectors (see, for example, EP-A-194809).

[0262] Preferably, the vector for use in plants includes border sequences that allow the expression cassette to be transferred and integrated 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 constructs include, for example, pBin19 (see Frisch D.A., L.W. Harris-Haller et al., 1995, the complete sequence of the binary vector Bin 19, Plant Molecular Biology, 27, 405-409).

[0263] Suitable promoters that operate in plants include Cauliflower Mosaic Virus 35S (CaMV 35S). Other examples are given in Lindsey and Jones, Plant Biotechnology in Agriculture (1989), page 120, published by OU Press, Milton Keynes, UK. The promoter may be selected to include one or more sequence motifs or elements that confer developmental and / or tissue-specific expression regulation. Inducible plant promoters include the ethanol-inducible promoter described by Caddick et al. (Nature Biotechnology, 1998, 16, 177-180).

[0264] If necessary, the construct may contain selectable genetic markers, such as genetic markers that confer selectable phenotypes such as resistance to antibiotics or herbicides (such as kanamycin, hygromycin, phosphinothricin, chlorsulfuron, methotrexate, gentamicin, spectinomycin, imidazolinone and glyphosate). Using a positive selection system (such as the positive selection system described by Haldrup et al. (Plant Molecular Biology, 1998, 37, 287-296)), constructs that do not rely on antibiotics can be obtained.

[0265] As mentioned above, one preferred vector is the "CPMV-HT" vector described in WO2009 / 087391. The following examples demonstrate the use of such a pEAQ-HT expression plasmid.

[0266] Such vectors (generally binary vectors) used in the present invention generally include an expression cassette comprising:

[0267] (i) a promoter operably linked to:

[0268] (ii) an enhancer sequence derived from the RNA-2 genomic segment of a bipartite RNA virus, wherein the target start site in the RNA-2 genomic segment has been mutated;

[0269] (iii) a nucleic acid sequence as described above;

[0270] (iv) a terminator sequence; and optionally,

[0271] (v) a 3'UTR located upstream of the terminator sequence.

[0272] Enhancer sequences (or enhancer elements) are sequences derived from the RNA-2 genomic fragment of a bipartite RNA virus such as cowpea mosaic virus (or sequences with homology to the genomic fragment), in which the target start site has been mutated. Such sequences can enhance the downstream expression of the heterologous open reading frame (ORF) to which they are attached. When present in a transcribed RNA, such sequences can also enhance the translation of the heterologous ORF to which they are attached.

[0273] The target start site refers to the start site (start codon) of the wild-type RNA-2 genomic fragment of the bipartite virus (such as cowpea mosaic virus) from which the target enhancer sequence is derived, which is used to produce (translate) the longer of the two proteins that share a carboxyl terminus encoded by the wild-type RNA-2 genomic fragment.

[0274] As mentioned above, the RNA virus is typically cowpea mosaic virus.

[0275] The most preferred vector is the pEAQ vector described in WO2009 / 087391, which enables direct cloning via a polylinker between the 5' leader sequence and the 3' UTR of the expression cassette containing the translation enhancer of the present invention and located on T-DNA, wherein the T-DNA further comprises a gene silencing suppressor and an NPTII box.

[0276] In such gene expression systems, gene silencing suppressors are preferably present, but not necessarily present. As described in WO / 2007 / 135480, there are known gene silencing suppressors in the art. Such gene silencing suppressors include HcPro from potato virus Y, He-Pro from TEV, P19 from TBSV, rgsCam and B2 proteins from FHV, small coat proteins from CPMV, and coat proteins from TCV. When producing stable transgenic plants, the preferred suppressor is a P19 suppressor containing an R43W mutation.

[0277] As described herein, a host can be converted from a phenotype that is incapable of efficient biosynthesis as described herein to a phenotype that is capable of such biosynthesis, such that the product can be recovered or used in vivo to synthesize downstream products.

[0278] The biosynthesis may include: (i) converting 2,3-epoxysqualene into intermediates such as squalane acid, oleanolic acid or echinocystic acid; (ii) converting squalane acid into intermediates such as QA-Tri, QA-Mono or QA-Di; (iii) converting QA-Tri into intermediates such as QA-TriFRXX or QA-TriF, QA-TriFR or QA-TriFX; (iv) converting squalane acid into intermediates such as SpB or QA-TriF(Q)RXX.

[0279] The biosynthesis may further include: (i) converting 2,3-epoxysqualene to β-amyrin; (ii) converting β-amyrin to oleanolic acid; (iii) converting oleanolic acid to echinocarpic acid; (iv) converting echinocarpic acid to sapotic acid; (v) converting sapotic acid to 3-O-{[β-D-pyranoglucuronic acid]oxy}-sapotic acid (QA-GlcA); (vi) converting 3-O-{[β-D-pyranoglucuronic acid]oxy}-sapotic acid (QA-GlcA) to 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-sapotic acid (QA-GlcA- Gal); (vii) conversion of 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA-Gal) into 3-O-{β-D-pyranoxylanosyl-(1→3)-[β-D-pyranoxylanosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA-Gal-Xyl or QA-Tri); (viii) conversion of QA-Tri into 3-O-{β-D-pyranoxylanosyl-(1→3)-[β-D-pyranoxylanosyl-(1→2)]-β-D-pyranoglucuronic acid}-28 -O-{β-D-fucopyranosyl ester}-saponic acid (QA-TriF); (ix) converting QA-TriF into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponic acid (QA-TriFR); (x) converting QA-TriFR into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28 (xi) converting QA-TriFRX into 3-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponic acid (QA-TriFRX); (xi) converting QA-TriFRX into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponic acid (QA-TriFRXX);(xii) converting QA-TriFRXX into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponic acid (QA-TriF(Q)RXX); and / or or (xii) converting QA-TriF(Q)RXX into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-2)-[β-D-4-O-pyranacetylquinovosyl-(1→4)]-β-D-fucopyranosyl ester}-saponic acid (SpB). ;

[0280] As described above, the triterpene biosynthesis genes described herein can also be modified in plants. Suitable techniques exist in the art (see, for example, WO2019 / 122259). 2,3-epoxysqualene is commonly found in higher plants due to its role in sterol biosynthesis, so the biosynthesis described herein can be widely used in plant hosts. Suitable plant hosts include any plant that is easily transformed with Agrobacterium. As described herein, when the methods described herein are actually used for microorganisms, other activities can also be further utilized.

[0281] Suitable hosts include plants such as Nicotiana benthamiana and microorganisms such as yeast. This will be described in further detail below.

[0282] The present invention may include: introducing the biosynthetic nucleic acid as described above into a host cell via a vector, transforming the host with the heterologous nucleic acid; and causing or allowing the vector to recombine with the genome of the host cell to introduce the nucleic acid of the present invention into the genome.

[0283] Another aspect of the present invention provides a host cell transformed with a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a plurality of sequences of triterpene biosynthetic nucleotides, each of which encodes a polypeptide, and wherein the triterpene biosynthetic nucleotides in combination have the biosynthetic activity described herein, wherein the expression of the nucleic acid enables the transformed host to have the ability to carry out the biosynthesis, or enhances the host's ability to do so.

[0284] The present invention also includes a host cell, particularly a plant or microbial cell, transformed with a triterpene biosynthetic nucleic acid or vector as described above (e.g., containing a biosynthetic modification nucleotide sequence). In the transgenic host cell (i.e., a transgenic host cell for the nucleic acid), the transgene may be on an extragenomic vector or contained in the genome, preferably stably contained in the genome. More than one heterologous nucleotide sequence may be present in each haploid genome.

[0285] The methods and materials described herein can be used for purposes such as producing stable crops that accumulate biosynthesized triterpenoid saponins or other products. Plants include, for example, row crops such as sunflower, potato, canola, dry beans, grain peas, flax, safflower, buckwheat, cotton, corn, soybeans, and sugar beets. In addition, major crops such as corn, wheat, oilseed rape, and rice can also be preferred hosts.

[0286] Furthermore, a plant comprising the plant cell of the present invention is also provided.

[0287] In addition, a method is also provided comprising the steps of: introducing such a construct into a plant cell or a microorganism (such as bacteria, yeast or fungus) cell; and / or inducing the expression of the construct in a plant cell by applying a suitable stimulus (such as an effective exogenous inducer).

[0288] As an alternative to microorganisms, cell suspension cultures of engineered plant species that produce glycosylated sapogenins (e.g., see Grotewold et al., Engineering secondary metabolites in maize cells by ectopic expression of transcription factors, Plant Cell, 1998, vol. 10, pp. 721-740) can be grown in fermenters, including the moss Physcomitrella patens.

[0289] In addition, a host cell, especially a plant or microbial cell, containing the above heterologous construct is also provided.

[0290] The above description of host cells related to reconstitution of quince or glycosylated quince biosynthesis in heterologous organisms also applies here.

[0291] In addition, a method for transforming plant cells is also provided, comprising: introducing the construct as described above into plant cells; and causing or allowing the vector and the genome of the plant cell to recombine to introduce the nucleic acid described herein into the genome.

[0292] The present invention also includes a host cell, especially a plant or microbial cell, transformed with a nucleic acid or vector as described herein (e.g., containing a triterpene biosynthetic nucleotide sequence). In the transgenic plant cell (i.e., a transgenic plant cell for the nucleic acid), the transgene may be on an extragenomic vector or contained in the genome, preferably stably contained in the genome. More than one heterologous nucleotide sequence may be present in each haploid genome.

[0293] Yeast has been widely used as a host for triterpene production and thus may be well suited for the biosynthesis of sapogenins and glycosylated sapogenins as described herein, such as the triterpene saponins.

[0294] In some preferred embodiments, the host is yeast. For such hosts, it may be necessary to increase the flux of saponic acid by introducing other genes, and thereby increase the production of saponic acid or glycosylated saponic acid as described above. Such other genes may, for example, include one or more plant cytochrome P450 reductases (CPRs) used as reduction partners of the introduced P450, and HMGR. Similarly, it may also be necessary to provide other components of saponic acid by introducing other genes, or to improve the saponic acid glycosylation pathway. Such other genes may include enzymes that provide UDP sugar donors, etc. (see, for example, Ohashi T, Hasegawa Y, Misaki R, Fujiyama R, Substrate preference of naringenin rhamnosyltransferase and its application in the production of flavonoid glycosides in fission yeast, 2016, Applied Microbiology and Biotechnology, Vol. 100, No. 2, pp. 687-696; Oka T, Jigami Y, Reconstruction of the de novo synthesis pathway of UDP-glucuronic acid and UDP-xylose from UDP-glucose in Saccharomyces cerevisiae, 2006, Journal of the Federation of European Biochemical Societies (FEBSJ.), Vol. 273, No. 12, pp. 2645-2657). According to the present disclosure, those skilled in the art can provide such additional activities as needed.

[0295] Furthermore, a plant comprising the plant cell transformed in the above manner is also provided.

[0296] After plant cell transformation, plants can be regenerated from single cells, callus or leaf discs, as required, according to standard practices in the art. Almost any plant can be completely regenerated from its cells, tissues and organs. Available techniques are summarized in the following works: Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, by Vasil et al., Academic Press, 1984; Methods for Plant Molecular Biology, by Weissbach and Weissbach, Academic Press, 1989.

[0297] In addition to regenerated plants, clones of such plants, seeds, self-fertilized or hybridized progeny and offspring (such as F1 and F2 offspring) are also provided. In addition, plant propagules derived from such plants are also provided, which are any parts that can be used for sexual or asexual reproduction or propagation, including cuttings, seeds, etc. In any case, such plants or parts include the above-mentioned plant cells or heterologous biosynthetic modified nucleic acids (for example, introduced into ancestral plants).

[0298] In addition, any part of such plants (such as leaves, stems, dried or ground products, edible parts, etc.) is also provided, which in any case includes the above-mentioned plant cells or heterologous triterpene biosynthetic DNA.

[0299] The present invention also includes the expression products of any nucleic acid sequence encoding triterpene biosynthesis disclosed herein, and correspondingly includes methods of producing the expression products by expressing the encoding nucleic acid under appropriate conditions, wherein the expression may occur in a suitable host cell.

[0300] As described below, the plant background (such as the plant background described above) can be a natural background, or a transgenic background, such as a transgenic background for one or more other genes related to the biosynthesis of triterpenes such as saponic acid or glycosylated saponic acid or otherwise affecting its phenotype or trait.

[0301] In modifying the host phenotype, the triterpene biosynthetic nucleic acids described herein can be used in combination with any other gene, such as a transgene that affects the biosynthesis rate or production of a triterpene such as sapogenin or glycosylated sapogenin, or a transgene that affects its modification or any other phenotypic trait or desired property.

[0302] By using a combination of genes, plants or microorganisms (such as bacteria, yeast or fungi) can be modified to enhance their ability to produce the desired precursors or to reduce their adverse metabolic effects.

[0303] A triterpene biosynthetic sequence described herein can be used to catalyze a corresponding biological activity in vitro or in vivo.

[0304] For example, a method for converting 2,3-epoxysqualene (OS) into β-amyrin may include: contacting 2,3-epoxysqualene with a saponin β-amyrin synthase (SobAS) containing the amino acid sequence of SEQ ID NO: 8 or a variant thereof, so that 2,3-epoxysqualene is converted into β-amyrin. In addition, the use of a saponin β-amyrin synthase (SobAS) containing the amino acid sequence of SEQ ID NO: 8 or a variant thereof to convert 2,3-epoxysqualene into β-amyrin is also provided.

[0305] A method for oxidizing the C-28 position of β-amyrin to carboxylic acid may include: contacting β-amyrin with a SoC28 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof, so that the C-28 position of β-amyrin is oxidized to a carboxylic acid, thereby generating oleanolic acid. In addition, the use of the SoC28 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof to oxidize the C-28 position of β-amyrin to a carboxylic acid is also provided.

[0306] A method for producing echinocystic acid by oxidizing the C-16 position of oleanolic acid to an alcohol may include: contacting oleanolic acid with a SoC28C16 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 4 or a variant thereof, so that the C-16 position of oleanolic acid is oxidized to an alcohol, thereby producing echinocystic acid. In addition, the use of a SoC28C16 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 4 or a variant thereof to produce echinocystic acid by oxidizing the C-16 position of oleanolic acid to an alcohol is also provided.

[0307] A method for oxidizing the C-16 position of β-amyrin to alcohol and the C-28 position to carboxylic acid to generate echinocystic acid may include: contacting β-amyrin with a SoC28C16 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 4 or a variant thereof, so that the C-16 position of β-amyrin is oxidized to alcohol and the C-28 position is oxidized to carboxylic acid, thereby generating echinocystic acid. In addition, the use of the SoC28C16 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 4 or a variant thereof to generate echinocystic acid by oxidizing the C-28 and C-16 positions of β-amyrin is also provided.

[0308] A method for generating quince acid (QA) by oxidizing the C-23 position of quince acid to an alcohol may include: contacting quince acid with a SoC23 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 6 or a variant thereof, so that the C-23 position of quince acid is oxidized to an aldehyde, thereby generating quince acid (QA). In addition, the use of a SoC23 oxidase polypeptide containing the amino acid sequence of SEQ ID NO: 6 or a variant thereof to generate quince acid (QA) by oxidizing the C-23 position of β-amyrin or an oxidized derivative thereof to an aldehyde is also provided.

[0309] A method for converting saponic acid (QA) into 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA) may include: contacting saponic acid with a Soapwort QA3-O glucuronyltransferase (SoCSL) polypeptide containing the amino acid sequence of SEQ ID NO: 10 or a variant thereof, so that QA is converted into QA-GlcA. In addition, the use of a SoCSL polypeptide containing the amino acid sequence of SEQ ID NO: 10 or a variant thereof to convert saponic acid into QA-GlcA is also provided.

[0310] A method for converting 3-O-{β-D-pyranoglucuronic acid}-saponinic acid (QA-GlcA) into 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponinic acid (QA-GlcA-Gal) may include: contacting QA-GlcA with a Saponinia sinensis QA-GlcA galactosyltransferase (SoC3Gal) polypeptide containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof, so that QA-GlcA is converted into QA-GlcA-Gal. In addition, use of a Saponinia sinensis QA-GlcA galactosyltransferase (SoC3Gal) polypeptide containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof to convert QA-GlcA into QA-GlcA-Gal is also provided.

[0311] A method for converting 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-glucuronic acid}-saponic acid (QA-GlcA-Gal) into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronic acid}-saponic acid (QA-GlcA-[Gal]-Xyl or QA-Tri) may include: contacting QA-GlcA with a Saponaria officinalis QA-GlcA-Gal xylosyltransferase (SoC3Xyl) polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof, so that QA-GlcA-Gal is converted into QA-Tri. In addition, the invention also provides the use of a soapwort QA-GlcA-Gal xylosyltransferase (SoC3Xyl) polypeptide containing the amino acid sequence of SEQ ID NO: 14 or a variant thereof to convert QA-GlcA-Gal into QA-Tri.

[0312] A method for converting QA-Tri into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF) may include: contacting QA-Tri with a Saponaria officinalis QA-Tri fucosyltransferase (SoC28Fu) polypeptide containing the amino acid sequence of SEQ ID NO: 16 or a variant thereof, so that QA-Tri is converted into QA-TriF. In addition, use of a Saponaria officinalis QA-Tri fucosyltransferase (SoC28Fu) polypeptide containing the amino acid sequence of SEQ ID NO: 16 or a variant thereof to convert QA-Tri into QA-TriF is also provided.

[0313] A method for converting QA-TriF into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFR) may include: contacting QA-TriF with a saponin QA-TriF rhamnosyltransferase (SoC28Rha) polypeptide containing the amino acid sequence of SEQ ID NO: 18 or a variant thereof, so that QA-TriF is converted into QA-TriFR. In addition, use of a saponin QA-TriF rhamnosyltransferase (SoC28Rha) polypeptide containing the amino acid sequence of SEQ ID NO: 18 or a variant thereof to convert QA-TriF into QA-TriFR is also provided.

[0314] A method for converting QA-TriFR into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRX) may include: contacting QA-TriFR with a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) polypeptide containing the amino acid sequence of SEQ ID NO: 20 or a variant thereof, so that QA-TriFR is converted into QA-TriFRX. In addition, use of a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) polypeptide containing the amino acid sequence of SEQ ID NO: 20 or a variant thereof to convert QA-TriFR into QA-TriFRX is also provided.

[0315] A method for converting QA-TriFRX into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRXX) may include: contacting QA-TriFRX with a Saponaria officinalis QA-TriFRX xylosyltransferase (SoC28Xyl2) polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof, so that QA-TriFRX is converted into QA-TriFRXX. In addition, the invention also provides the use of a soapwort QA-TriFRX xylosyltransferase (SoC28Xyl2) containing the amino acid sequence of SEQ ID NO: 22 or a variant thereof to convert QA-TriFRX into QA-TriFRXX.

[0316] A method for converting QA-TriFRXX into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin (QA-TriF(Q)RXX) may include: contacting QA-TriFRXX with a Saponaria officinalis QA-TriFRXX quinovosyltransferase (SoGH1) polypeptide comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, so that QA-TriFRXX is converted into QA-TriF(Q)RXX. In addition, the invention also provides the use of a soapwort QA-TriFRXX quinoside transferase (SoC28Xyl2) containing the amino acid sequence of SEQ ID NO: 34 or a variant thereof to convert QA-TriFRXX into QA-TriF(Q)RXX.

[0317] A method for converting QA-TriF(Q)RXX into 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-2)-[β-D-4-O-pyranacetylquinovosyl-(1→4)]-β-D-fucopyranosyl ester}-saponic acid (QA-TriF(Q-Ac)RXX) may include: reacting QA-TriF(Q)RXX with a mixture containing SEQ ID NO:36 amino acid sequence or a variant thereof, so that QA-TriF(Q)RXX is converted into QA-TriF(Q-Ac)RXX. In addition, the use of the soapwort QA-TriF(Q)RXX acetyltransferase (SoBAHD1) containing the amino acid sequence of SEQ ID NO:36 or a variant thereof to convert QA-TriF(Q)RXX into QA-TriF(Q-Ac)RXX(SpB) is also provided.

[0318] In some embodiments, the one or more nucleic acids or proteins described above can be used for heterologous reconstruction of a biosynthetic pathway. The biosynthetic pathway is the above pathway and can include one or more of the conversion of 2,3-epoxysqualene to squalic acid, the conversion of squalic acid to QA-Tri, the conversion of QA-Tri to QA-TriFRXX, and the conversion of QA-TriFRXX to QA-TriF(Q-Ac)RXX.

[0319] In addition, a method for influencing or acting on biosynthesis in a host such as a plant is further provided, the method comprising: causing or allowing the heterologous triterpene biosynthetic nucleic acid as described above to be transcribed in a plant cell. Prior to this step, a prior step of introducing the nucleic acid into a plant or its ancestor cell may be performed. The biosynthesis may include: generating sapotic acid, glycosylated sapotic acid such as QA-Tri, QA-TriFRXX or QA-TriF(Q-Ac)RXX, or an intermediate of any one of them.

[0320] Such methods generally constitute a part of (and possibly a step of) a method for producing glycosylated saponin (such as QA-Tri, QA-TriFRXX or QA-TriF(Q-Ac)RXX) in a host such as a plant. Preferably, the method employs a triterpene biosynthetic polypeptide or a variant thereof as described above, or a nucleic acid encoding any one of them.

[0321] The above method can be used to produce saponic acid or glycosylated saponic acid such as QA-Tri, QA-TriFRXX or QA-TriF(Q-Ac)RXX in a heterologous host, or can be used to produce intermediates. Glycosylated saponic acid is generally a non-natural substance in the species to be introduced.

[0322] Triterpenes obtained from the plants or methods described herein, including glycosylated forms of quince acid such as QA-Tri, QA-TriFRXX or QA-TriF(Q-Ac)RXX, can be isolated and used commercially.

[0323] The above method may constitute part of (and possibly a step of) a method for producing downstream products such as QS-21 in a host. The method may include the steps of culturing the host (when the host is a microorganism), or growing the host (when the host is a plant); and then harvesting the host, and purifying triterpenes such as glycosylated saponins such as QA-Tri, QA-TriFRXX or QA-TriF(Q-Ac)RXX, or downstream products or derivatives (such as QS-21) obtained from the host. The products produced in this manner constitute another aspect of the present invention. The functions of QS-21 are described above.

[0324] As an alternative, glycosylated quince acids such as QA-Tri, QA-TriFRXX, QA-TriF(Q-Ac)RXX can be recovered for further chemical synthesis of downstream compounds.

[0325] The methods described herein include both in vitro and in vivo generation or manipulation of triterpenes such as sapogenin and / or one or more glycosylated sapogenin. For example, triterpene biosynthetic polypeptides can be used for fermentation by expression in microorganisms, such as Escherichia coli, yeast, and filamentous fungi. In some embodiments, one or more newly characterized triterpene biosynthetic sequences described herein can be used in combination with one or more other biosynthetic genes in such organisms.

[0326] In vivo methods have been described above in large numbers and generally include the steps of causing or allowing transcription of a recombinant nucleic acid molecule encoding a triterpene biosynthetic polypeptide and then causing or allowing translation from the recombinant nucleic acid molecule.

[0327] In other embodiments, the triterpene biosynthetic polypeptide (enzyme) can be used in vitro, for example, in an isolated, purified or semi-purified form. Alternatively, such triterpene biosynthetic polypeptide (enzyme) can be the expression product of a recombinant nucleic acid molecule.

[0328] In order to reduce the adverse metabolic effects or fluxes that may affect the production of triterpenes such as sapogenin or glycosylated sapogenin, it may be necessary to down-regulate the genes in the host. Such down-regulation can be achieved by known methods in the art, for example, by antisense technology.

[0329] When antisense genes or incomplete gene sequences are used to downregulate gene expression, the nucleotide sequence is placed under the control of the promoter in the "reverse orientation" so that the RNA produced by transcription is complementary to the normal mRNA transcribed from the "sense" strand of the target gene, see, for example: Rothstein et al., 1987; Smith et al., Nature, 1998, Vol. 334, pp. 724-726; Zhang et al., Plant Cell, 1992, Vol. 4, pp. 1575-1588; English et al., Plant Cell, 1996, Vol. 8, pp. 179-188. For antisense technology, see: Bourque, Plant Science, 1995, Vol. 105, pp. 125-149; Flavell, PNAS USA, 1994, Vol. 91, pp. 3490-3496.

[0330] As an alternative to antisense technology, a copy of the entire target gene or a portion thereof can be inserted into the sense strand (i.e., in the same direction as the target gene) to reduce the expression of the target gene through co-suppression, for example, see: van der Krol et al., The Plant Cell, 1990, Vol. 2, pp. 291-299; Napoli et al., 1990, No. 2, pp. 279-289; Zhang et al., The Plant Cell, 1992, Vol. 4, pp. 1575-1588; and USA5231020. Further improvements in gene silencing or co-suppression technology can be found in: WO95 / 34668 (Biosource); Angell and Baulcombe, The EMBO Journal, 1997, Vol. 16, No. 12, pp. 3675-3684; and Voinnet and Baulcombe, Nature, 1997, Vol. 389, p. 553.

[0331] It has been found that double-stranded RNA (dsRNA) is even more effective in gene silencing than using the sense strand or antisense strand alone (Fire A, Nature, 1998, Vol. 391). Silencing mediated by dsRNA is a gene-specific type of silencing, commonly known as RNA interference (RNAi) (see further: Fire, Trends Genet., 1999, Vol. 15, pp. 358-363; Sharp, Genes Dev., 2001, Vol. 15, pp. 485-490; Hammond et al., Nature Rev. Genes, 2001, Vol. 2, pp. 1110-1119; and Tuschl, Chem. Biochem., 2001, Vol. 2, pp. 239-245).

[0332] RNA interference is a two-step process. First, inside the cell, dsRNA is cut into short interfering RNAs (siRNAs) of approximately 21 to 23 nucleotides in length, with a phosphate group at the 5' end and a short overhang (approximately 2 nucleotides) at the 3' end. siRNA specifically targets the corresponding mRNA sequence for destruction (Zamore, P.D., Nature Structural Biology, 2001, Vol. 8, No. 9, pp. 746-750).

[0333] In another method known in the art to downregulate a target sequence, "microRNA" (miRNA) is used, see, for example, Schwab et al., Plant Cell, 2006, vol. 18, pp. 1121-1133. This technique uses artificial miRNAs, which can be encoded by stem-loop precursors containing appropriate oligonucleotide sequences, which can be generated according to the present disclosure and according to well-defined rules.

[0334] In some embodiments, a method for influencing or acting on the biosynthesis of sapogenin or glycosylated sapogenin in a host comprises any of the following steps:

[0335] (i) causing or allowing transcription from a nucleic acid comprising a complementary sequence to a host nucleotide sequence as described herein, so that the activity of the corresponding encoded polypeptide is attenuated by an antisense mechanism;

[0336] (ii) causing or allowing transcription from a nucleic acid encoding a stem-loop precursor of 20 to 25 nucleotides and optionally including one or more mismatches of a host nucleotide sequence, so that the activity of the corresponding encoded polypeptide is attenuated by the miRNA mechanism;

[0337] (iii) causing or allowing transcription from a nucleic acid encoding a double-stranded RNA corresponding to 20 to 25 nucleotides of the host nucleotide sequence and optionally including one or more mismatches, such that the activity of the corresponding encoded polypeptide is attenuated by the siRNA mechanism.

[0338] It will be appreciated by those skilled in the art that, in light of this disclosure, other genes may be used in the practice of the present invention to provide additional activity and / or enhance expression or activity. Such other genes include genes that express cofactors or auxiliary proteins, or proteins that express other factors.

[0339] It should be understood that when the above general terms are used in conjunction with any aspect or embodiment, their meanings or disclosures are deemed to be equally applicable to any of the above sequences, respectively.

[0340] Aspects and embodiments obtained by replacing the word "comprising" in the above aspects and embodiments with "consisting of..." and replacing the word "comprising" in the above aspects and embodiments with "consisting essentially of..." constitute other aspects and embodiments of the present invention.

[0341] It should be understood that, unless the context otherwise requires, this application discloses all combinations of any of the above aspects and the above embodiments with each other. Similarly, unless the context otherwise requires, this application discloses all combinations of preferred and / or optional features used alone or in combination with any other aspect.

[0342] Modifications of the above-described embodiments, other embodiments, and modifications of other embodiments will become apparent to those skilled in the art after reading this disclosure, and such modes and schemes are also within the scope of the present invention.

[0343] All documents and sequence database entries mentioned in this specification are hereby incorporated by reference in their entirety for all purposes.

[0344] The term "and / or" as used herein shall be deemed to specifically disclose each of the situations where only one of the preceding and following features or components is present or both of them are present. For example, "A and / or B" shall be deemed to specifically disclose the following three situations: (i) "there is A"; (ii) "there is B"; and (iii) "there are A and B", and is equivalent to describing each of the situations in this document.

[0345] abbreviation

[0346] QA-GlcA-[Gal]-Xyl or QA-Tri: 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin

[0347] QA-GlcA-Gal or QA-Di: 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin

[0348] QA-GlcA or QA-MonO: 3-O-{β-D-pyranoglucuronic acid}-saponic acid

[0349] QA-TriF: 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin

[0350] QA-TriFR: 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin

[0351] QA-TriFRX: 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin

[0352] QA-TriFRXX: 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1→3)-β-D-pyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin

[0353] QA-TriF(Q)RXX: 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin

[0354] QA-TriF (Q-Ac) RXX, SpB or saponin B: 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-2)-[β-D-4-O-pyranacetylquinovosyl-(1→4)]-β-D-fucopyranosyl}-saponin

[0355] QA: Soap acid

[0356] OS: 2,3-Epoxysqualene

[0357] Gal: D-galactopyranose

[0358] GlcA: D-pyranoglucuronic acid (the additional number indicates the specific carbon atom, such as GlcA-1)

[0359] Xyl:D-xylopyranose

[0360] Rha: L-rhamnose

[0361] Ac:Acetyl

[0362] Qui: D-Quinol (or Q)

[0363] SobAS or SobAS1: saponaria β-amyrin synthase

[0364] SoC28 oxidase, SoC28 or CYP716A378: Soapwort saponin C28 oxidase

[0365] SoC16 oxidase, SoC28C16 oxidase, SoC28C16 or CYP716A379: Soapwort saponin C28 and C16 oxidase

[0366] SoC23 oxidase, SoC23 or CYP72A984: Soapwort saponin C23 oxidase

[0367] SoQA-GlcAT, SoCSL or SoCSL1: Soapberry salsa 3-O glucuronyltransferase

[0368] SoQA-GalT, SoC3Gal or UGT73DL1: Saponaria officinalis QA-GlcA galactosyltransferase

[0369] SoQA-XylT, SoC3Xyl or UGT3CC6: Saponaria officinalis QA-GlcA-Gal xylosyltransferase

[0370] SoQA-TriFuT, SoC28Fu or UGT74CD1: Saponaria officinalis QA-Tri fucosyltransferase

[0371] SoFuSyn or SoSDR: Saponaria short-chain dehydrogenase

[0372] SoQA-TriFRhaT, SoC28Rha or UGT79T1: Saponaria officinalis QA-TriF rhamnosyltransferase

[0373] SoQA-TriFRXylT, SoC28Xyl1 or UGT79L3: Saponaria officinalis QA-TriFR xylosyltransferase

[0374] SoQA-TriFRXXylT, SoC28Xyl2 or UGT73M2: Saponaria officinalis QA-TriFRX xylosyltransferase

[0375] SoGH1: Saponaria officinalis QA-TriFRXX quinolysate transferase

[0376] SoBAHD1: Saponaria officinalis QA-TriF(Q)RXX acetyltransferase

[0377] tHMGR: Truncated 3-hydroxy-3-methylbutyryl-CoA reductase from Avena strigosa (diploid oat)

[0378] experiment

[0379] Materials and methods

[0380] RNA synthesis and RNA sequence analysis

[0381] Total RNA was extracted from roots and leaves of representative Sophora japonica plants using the RNeasy Plant Mini Kit following a modified protocol described by MacKenzie et al. (Plant Disease, 1997, vol. 81, pp. 222-226). In addition to RNA extraction, on-column DNase digestion was performed using RNase-free DNase (Promega). The GoScript TM Reverse transcriptase (Promega) was used according to the manufacturer's instructions to generate cDNA for amplification of putative P. basalticum baS using 0.8 μg of DNase-treated RNA.

[0382] A total of 24 RNA samples were sent to the Earlham Institute (EI) for transcriptome sequencing and RNA sequence analysis. Among them, NEBNext Ultra II directional RNA sequence libraries were constructed with 24 RNA samples and sequenced in two lanes of the NovaSeq 6000SP flow cell (150bp double-end reads). The Earlham Institute used Trinity de novo assembly tool (version 2.8.5) for transcriptome assembly, and TransDecoder (version 5.5.0) and Human Readable Descriptions (AHRD, version 3.3.3) for ORF prediction and functional annotation, respectively. The Earlham Institute also used salmon (version 0.14.1) for transcript quantification.

[0383] Candidate gene identification

[0384] To identify candidate bASs in Sophora flavescens, the Sophora flavescens transcriptome was obtained from the One Thousand Plants (1KP) project (www.onekp.com, Wicket et al., Proceedings of the National Academy of Sciences of the United States of America, 2014, vol. 45, pp. 4859-4868). A BLASTP search was performed against the translated Sophora flavescens protein database using the previously characterized OSCs from other plant species listed in Table 1 as queries. The Sophora flavescens candidate gene list was first screened by eliminating sequences less than 500 amino acids (aa) in length and then further screened by phylogenetic analysis using MEGA-X (http: / / www.megasoftware.net). The putative basidiomycete genes were aligned with the published OSCs from other plants listed in Table 1 using the MUSCLE algorithm (https: / / www.ebi.ac.uk / Tools / msa / muscle / ) for amino acids, and a phylogenetic tree was created using the alignment results, using a neighbor-joining algorithm (Poisson model) with 1000 bootstrap replicates. Based on the results of the phylogenetic analysis, candidate genes that were unlikely to be bAS were eliminated from the list.

[0385] After identifying SobAS, all other pathway candidate genes were further identified using the newly assembled Saponaria officinalis transcriptome from the Earlham Institute. A BLASTP search was performed on the new Saponaria officinalis transcriptome using published gene families as queries to create preliminary lists of candidate Saponaria officinalis CYP450s, CSLs, and UGTs, respectively. These lists were first screened by eliminating candidate genes with a length of less than 500 aa and then further refined by correlation analysis to identify candidate genes with similar expression patterns to SobAS. All bioinformatics analyses were performed using R; transcript quantification results for salmon were read in using tximport (version 1.18.0); regularized logarithmic (rlog) library normalized reads were generated using DEseq2 (version 1.30.1) and hierarchical clustering was performed; correlation analysis was performed using the Pearson method.

[0386] cDNA synthesis and clone

[0387] To clone candidate P. oleracea genes, cDNA pools were generated from root and leaf RNA. First-strand cDNA synthesis was performed using the GoScript reverse transcription system (Promega) according to the manufacturer's protocol.

[0388] For candidate phytophthora genes except SoGH1, their coding sequences were PCR amplified from the cDNA pool using gene-specific primers with 5'AttB sites. For SoGH1 with 5'AttB sites, its coding sequence was synthesized by IDT. PCR products were purified using QIAquick PCR purification kit according to the manufacturer's protocol. The pDONR207 vector was transferred to the entry vector by the technology (Invitrogen), and finally to the expression vector. In brief, the pDONR207 vector and the purified PCR product were first subjected to BP recombination reaction in equal proportions (about 150 ng each) according to the manufacturer's instructions, and then transformed into chemically competent Escherichia coli cells (DH5α, ThermoFisher Scientific) by heat shock. After preparing and recovering the plasmid with the QIAprep centrifugal miniprep kit (Qiagen), the sequence of the plasmid was verified. In order to generate expression clones, the entry vector carrying the target gene and the pEAQ-HT-DEST1 expression vector were subjected to LR recombination reaction in equal proportions (about 150 ng each) according to the manufacturer's protocol (Sainsbury et al., Journal of Plant Biotechnology, 2009, Vol. 7, No. 7, pp. 682-693). Subsequently, the QIAprep centrifugal miniprep kit (Qiagen) was used again and the plasmid was recovered according to the manufacturer's protocol.

[0389] Transient expression of candidate genes in Nicotiana benthamiana

[0390] After transient expression of the candidate gene in Nicotiana benthamiana using the Agrobacterium tumefaciens strain LBA4404 (Invitrogen), Agrobacterium infiltration, sample harvesting and preparation were performed according to the methods previously described by Reed et al. (Metabolic Engineering, 2017, Issue 42, Pages 185-193).

[0391] GC / MS analysis

[0392] GC / MS analysis was performed using an Agilent 7890B equipped with a Zebron Ab5 Ht Inferno column (Phenomenex) and following a 20-minute method developed by James Reed (Osbourn Laboratory). Briefly, each sample was injected at the inlet (250°C) in pulsed splitless mode (pulse pressure 30 psi) with an injection volume of 1 μL. The oven temperature was first maintained at 170°C for 2 minutes, then increased to 300°C at a rate of 20°C / min and maintained at 300°C for 11.5 minutes, so the total run time was 20 minutes. Mass spectra were performed with an Agilent 5977A mass selective detector in scan mode with a scan range of 60 to 800 m / z and a solvent delay time of 8 minutes. The data were analyzed with a MassHunter workstation (Agilent).

[0393] LC / MS analysis

[0394] LC / MS analysis was performed using a Shimadzu Prominence HPLC system equipped with an IT-TOF mass spectrometer (Shimadzu), wherein aqueous formic acid (0.1% v / v) was used as solvent A and acetonitrile was used as solvent B. Each sample was analyzed using a Kinetex XB-C18100A (50×2.1 mm, 2.6 μm; Finominis) column at a flow rate of 0.5 mL / min, a temperature of 40°C, and an injection volume of 5 μL. The mass spectrometer was equipped with electrospray in negative ionization mode (capillary temperature of 250°C, nebulizer gas flow rate of 1.3 min / L, heating block temperature of 300°C, and spray voltage of -3.5 kV). The elution scheme was as follows: 0-1 min, 5% solvent B in solvent A; 1-10 min, 55% solvent B in solvent A; 10-12 min, 100% solvent B; 12-13 min, 100% solvent B; 13-13.1 min, 5% solvent B in solvent A; 13.1-15.6 min, 5% solvent B in solvent A. The formation of product ions was monitored by MS / MS. Data acquisition and processing used LCMSolution software (Shimadzu). All saponin pathway intermediate standards were provided by members of the Osborne group.

[0395] The formation and transformation of hairy roots of Saponaria officinalis

[0396] Soapwort seeds were collected from plants grown in the JIC greenhouse. After washing with sterile water, the seeds were placed in sterile water for 3-4 hours, surface sterilized with sodium hypochlorite (5% w / v) for 30 minutes, and then washed three times with sterile water. In addition, the seeds were further washed with 70% ethanol (v / v) for 1 minute and then washed three times with sterile water. The seeds were germinated in MS (Murashige-Skoog, 1962) medium (pH = 5.88) containing 3% sucrose and 0.8% agar. After 4 weeks, the seedlings were subcultured and placed in MS medium (pH = 5.88) containing 3% sucrose and 0.8% agar at 25°C with a 16-hour light cycle. Hairy roots were induced with ATCC15834, which was found to be effective (100% induction) against other strains tested (A4, A4RS and LBA1334). Briefly, the corresponding bacterial solution (100 μM acetosyringone in MS medium containing 1% sucrose, optical density (OD) = 0.6) was injected into leaf explants by needle, and each leaf explant was injected about 5 times. The infected explants were placed in the dark at 25°C for 4 days for co-cultivation, which consisted of semi-solid (0.8% agar) MS medium supplemented with 3% sucrose and 100 μM acetosyringone. In addition, the explants were transferred to semi-solid (0.8% agar) MS medium supplemented with 3% sucrose, 500 mg / l cefotaxime and 50 mg / l kanamycin, and continued to be placed at 25°C with a 16-hour light cycle until the bacteria were eliminated and the desired hairy roots appeared.

[0397] After designing silencing primers based on regions unique to the saponaria β-amyrin synthase (SoβAS), it was cloned into pDONR207 (Gateway compatible vector). In addition, subcloning was performed in pK7WGIGW-2R to achieve dsRNA-mediated transgene silencing. To achieve overexpression of SoβAS, the full-length sequence was cloned into pK7WG2R using Gateway technology. In addition, control hairy roots were cultured using empty pK7WG2R (Zhao et al., 2016). All constructs were transformed into ATCC15834, and the transgenic status of hairy roots was evaluated by dsRed fluorescent protein and PCR after co-cultivation with wounded leaves. In addition, three-week-old dsRed-expressing hairy roots grown in B5 liquid medium (containing vitamins and sucrose) in the dark were evaluated for metabolite analysis.

[0398] result

[0399] Phylogeny-based identification and characterization of SobAS

[0400] It is speculated that the key steps in triterpene biosynthesis are the production of β-amyrin catalyzed by epoxysqualene cyclase (OSC) and β-amyrin synthase (bAS). To identify candidate bAS in Sophora flavescens, the translated Sophora flavescens transcriptome from the Thousand Plants (1KP) project (www.onekp.com, Wicket et al., supra) was mined and a reciprocal BLASTP search was performed using previously characterized OSCs from other plant species as search queries (Table 1). SobAS was identified as a possible candidate gene for Sophora flavescens bAS through phylogenetic analysis.

[0401] To test the activity of SobAS, SobAS was transiently expressed in Nicotiana benthamiana containing a truncated HMG-CoA reductase (tHMGR) to increase the flux to the MVA pathway (Reed et al., 2017, supra). The complete open reading frames of SobAS and tHMGR were transformed into Agrobacterium tumefaciens and then introduced into Nicotiana benthamiana leaves by co-infiltration. Four days after infiltration, the infiltrated leaves were harvested, their metabolites were extracted, and these metabolites were analyzed by GC / MS. The transient expression of SobAS in Nicotiana benthamiana resulted in the formation of peak 1 at m / z = 498, and the retention time and mass spectrum of this peak corresponded to the commercially available β-amyrin standard ( Figure 4 a). The negative control leaves expressing only tHMGR did not form peak 1 ( Figure 4 a). Based on this result, the candidate gene SobAS was identified as the OSC that can cyclize epoxysqualene to β-amyrin ( Figure 4 b).

[0402] Identification of saponin pathway genes by co-expression analysis

[0403] Since the publicly available transcriptome of Sophora japonica from the 1KP project does not contain any organ-specific transcriptome data, RNA sequence analysis was performed on six different Sophora japonica organs (flowers, flower buds, young leaves, old leaves, stems, and roots) with different saponin contents, and further gene identification was performed using the newly obtained Sophora japonica transcriptome rather than the transcriptome of the 1KP project.

[0404] It is speculated that after the biosynthesis of β-amyrin, the next step in the biosynthesis of saponins is the oxidation of β-amyrin to saponin acid by three cytochrome P450s (CYP450s). To create a list of candidate CYP450s for Sophora flavescens, a BLASTP search was performed on the newly assembled Sophora flavescens transcriptome using the TriForC database (http: / / bioinformatics.psb.ugent.be / triforc / , Miettinen et al., Nature Comms, 2017, Vol. 8, No. 1, pp. 1-13) as the query condition. This list was refined by eliminating all candidate genes with a length of less than 500 aa and further refined by performing Pearson co-expression analysis with the SobAS expression pattern characterized in the above manner. In the Pearson co-expression analysis, all candidate genes with a Pearson correlation coefficient (PCC) less than 0.80 in the candidate list were eliminated (Table 3).

[0405] It is speculated that the next step in the saponin biosynthesis pathway is to modify saponin acid with UDP-dependent glycosyltransferase (UGT) family 1. In order to identify candidate UGTs in Sophora flavescens, a list of previously identified UGTs from other plant species was first obtained from the following article: Louveau et al., Cold Spring Harbor Perspectives in Biology, 2019, Vol. 11, No. 12, a034744), and then a BLASTP search was performed on the Sophora flavescens transcriptome using these UGTs as query conditions. The candidate gene list was further refined in a similar manner to the above. Among them, Pearson co-expression analysis was performed with the SobAS expression pattern, and all candidate genes with a PCC value of less than 0.90 in the list were eliminated (Table 4).

[0406] In addition to UGTs, recent research results from Jozwiak and colleagues and members of the Osborne group have shown that cellulose synthase-like (CSL) genes have the ability to glucuronidate triterpenoid saponins (Jozwiak et al., 2020; WO / 2020 / 260475). For this reason, the transcriptome of the solitary solitary was also searched for candidate CSLs. Among them, a list of CSLs from other plant species that have been published in the literature was first obtained from Reed et al. (still in progress), and a BLASTP search was performed on the solitary solitary transcriptome using these CSLs as query conditions. The list of candidate solitary solitary CSLs was further refined by Pearson co-expression analysis based on the SobAS expression pattern, and all candidate solitary solitary CSLs in the list with a PCC value less than 0.85 were eliminated (Table 5). All putative saponin biosynthesis genes identified have similar expression patterns in different solitary solitary organs, indicating that they are involved in the same biosynthetic pathway ( Figure 3 ).

[0407] The candidate gene list was further screened and refined based on the degree of co-expression with the SobAS1 bait gene (sorted by PCC, selecting the one with higher PCC>0.88), annotation, and the absolute number of transcripts in the floral organ (selecting the one with higher PCC).

[0408] Characterization of candidate genes by transient expression in Nicotiana benthamiana

[0409] To test the activity of the candidate saponin biosynthesis genes identified above, they were transiently expressed in Nicotiana benthamiana. The open reading frames (ORFs) of the candidate genes were PCR amplified using the primers listed in Table 2 or synthesized with an upstream 5' attb site to allow for Cloning. Amplified or synthesized gene fragments were cloned into pDONR207 and transferred into the pEAQ-HT-DEST1 plant expression vector (Sainsbury et al., 2009, supra). Each expression construct was transformed into Agrobacterium tumefaciens (LBA4404) for transient expression in Nicotiana benthamiana. In all experiments, an Agrobacterium tumefaciens strain carrying tHMGR was also co-infiltrated to enhance triterpene production in Nicotiana benthamiana. By comparing Tables 3 to 5 and Figure 3 The activities of candidate genes ranked high in the list were screened, and SoC28, SoC28C16, SoC23, SoCSL, SoC3Gal, SoC3Xyl, SoC28Fu, SoC28Rha, SoC28Xyl1, SoC28Xyl2, SoGH1 and SoBAHD1 were identified.

[0410] To test the activity of SoC28 and SoC28C16, Agrobacterium tumefaciens strains carrying the ORFs of (i) tHMGR+SobAS+SoC28 or (ii) tHMGR+SobAS+SoC28C16 were co-infiltrated into N. benthamiana leaves. Four days after infiltration, leaves were harvested, metabolites were extracted, and metabolite analysis was performed by GC / MS. Co-expression of SobAS with SoC28 in N. benthamiana resulted in the formation of peak 2 at m / z=585 ( Figure 5 b). The retention time (RT), m / z and mass spectrum of peak 2 of the Nicotiana benthamiana extract correspond to peak 2 of the commercially available oleanolic acid standard, so peak 2 was identified as oleanolic acid ( Figure 5 b). Interestingly, in addition to oleanolic acid, another metabolite peak (peak 3) at m / z=570 was present in the extract of Nicotiana benthamiana leaves after co-infiltration of SobAS and SoC28C16. The RT and mass spectrum of peak 3 of the Nicotiana benthamiana extract corresponded to peak 3 of the echinococcal acid standard, so peak 3 was identified as echinococcal acid. Peaks 2 and 3 were not detected in the negative control Nicotiana benthamiana leaves expressing only tHMGR. Based on these results, SoC28 may be a CYP450 with C28 oxidation activity, thereby forming oleanolic acid from β-amyrin; SoC28C16 may be a CYP450 with both C28 and C16 oxidation activities, thereby forming oleanolic acid and echinococcal acid at the same time.

[0411] To test the activity of SoC23, Agrobacterium tumefaciens strains carrying OFRs of tHMGR+SobAS+SoC28C16+SoC23 were co-infiltrated into Nicotiana benthamiana leaves. After the leaves were harvested, their extracts were analyzed by HPLC / MS in negative ionization mode. The expression of SoC23 resulted in the formation of peak 4 at m / z=485.3, which corresponds to the [MH] - ( Figure 5 b). In addition, the retention time and mass spectrum of peak 4 match those of the sapotic acid standard. Peak 4 was not detected in the negative control Nicotiana benthamiana leaves expressing only tHMGR. Based on this result, SoC23 may be a CYP450 with C-23 oxidation activity.

[0412] After biosynthesis of saponic acid using genes from Saponaria officinalis, candidate SoCSL was co-expressed with genes required for saponic acid production (tHMGR+SobAS+SoC28C16+SoC23). After the leaves were harvested, their extracts were analyzed by HPLC / MS in negative ionization mode. HPLC / MS analysis showed that by adding SoCSL, peak 5 was formed at m / z=661.3, which was consistent with the expected [MH] of QA-Mono. - Peak coincidence ( Figure 6Peak 5 was not detected in the negative control expressing only tHMGR, and the RT and mass spectrum of Peak 5 were consistent with those of the QA-Mono standard obtained from the Osborne group ( Figure 6 The MS / MS fragmentation pattern of peak 5 also showed that the major fragment ion had an m / z of 485.33, which was consistent with the expected [MH] - Based on the above results, Peak 5 was identified as QA-Mono, and SoCSL was a CSL capable of glucuronidating quince acid.

[0413] Subsequently, the candidate SoC3Gal was co-expressed with genes required for the production of saponin and the newly characterized SoCSL (tHMGR+SobAS+SoC28C16+SoC23). Similar to the above, the leaves were harvested and their extracts were analyzed by HPLC / MS. Among them, the plant extract expressing only the genes for the production of saponin and SoCSL was used as a negative control. By adding SoC3Gal, a new peak was generated at m / z=823.4, which was consistent with the [MH] of QA-Di. - Corresponding ( Figure 7 ). In addition, the RT and mass spectrum of peak 6 generated by SoC3Gal match those of the QA-Di standard. In addition, the MS / MS fragmentation profile shows that the main fragment ion of peak 6 has an m / z of 485.32, which is consistent with the [MH] - This indicates that the sugar chains of QA-Di are fragmented ( Figure 7 ). Based on these results, Figure 7 Peak 6 in the above analysis was inferred to be QA-Di, and SoC3Gal was a galactosyltransferase derived from Saponaria officinalis.

[0414] After this, the candidate SoC3Xyl was characterized. The genes required for the production of QA-Di (tHMGR+SobAS+SoC28C16+SoC23+SoCSL+SoC3Gal) were co-expressed with the additional SoC3Xyl in N. benthamiana. After the leaves were harvested, their extracts were analyzed by HPLC / MS to detect the expected mass of m / z=955.4 and the [MH] - The negative control, which only co-expressed the genes required for the production of QA-Di, did not produce any peak at the expected m / z, but a new peak appeared at m / z = 955.4 due to the additional expression of SoC3Xyl ( Figure 8 Peak 7 has the same RT and mass spectrum as the QA-Tri standard, and the MS / MS fragmentation pattern shows that the major ion is [MH-Xyl] at m / z = 823.42. - and [MH-Xyl-Gal] at m / z = 485.33- ( Figure 8 ). Based on these results, it is inferred that Figure 8 Peak 7 in the figure is QA-Tri, and the SoC3Xyl candidate gene is xylose transferase.

[0415] The focus then was on characterizing sugar transferases with the activity of transferring D-fucose to QA-Tri. In previous studies, the Osborne group had identified two genes, QsC28Fu and QsFuSyn, that are involved in adding D-fucose to the QS-21 biosynthetic pathway. It has been shown that QsC28Fu has UDP-4-keto-6-deoxyglucose transferase activity, while QsFuSyn is a 4-ketoreductase (Reed, Orme, El-Demerdash et al., 2023). During this discovery process, SoFuSyn was also identified and characterized as having the activity of converting UPD-4-keto-6-deoxyglucose to UPD-D-fucose. The SoC28Fu candidate gene was identified by co-expression analysis with SobAS. In addition, the activity of the candidate gene was tested by transient expression in Nicotiana benthamiana. Among them, the genes required for the production of QA-Tri (tHMGR+SobAS+SoC28C16+SoC23+SoCSL+SoC3Gal+SoC3Xyl) were combined with the additional candidate SoC28Fu and the previously identified SoFuSyn and co-expressed in Nicotiana benthamiana. After the leaves were harvested, leaf extraction was performed and analyzed by HPLC / MS. The additional activity of SoC28Fu generated peak 8 (m / z=1101.5), which corresponded to the RT and mass spectrum of the peak generated by the QA-TriF standard authentic ( Fig. 9 This peak was not observed in the negative control without SoFuC28 ( Fig. 9 ). In addition, the MS / MS fragmentation pattern of peak 8 showed that the main product ion was: m / z = 955.4, which is the expected [MH] of QA-Tri - ; and m / z = 485.3. That is, [MH] of sap acid - ( Fig. 9 ). These results suggest that the candidate SoC28Fu can work together with SoFuSyn to transfer the fucose moiety to QA-Tri.

[0416] After this, the activity of the candidate SoC28Rha was detected. The genes required for the production of QA-TriF (tHMGR+SobAS+SoC28C16+SoC23+SoCSL+SoC3Gal+SoC3Xyl+SoFuSyn+SoC28Fu) were combined with the additionally added SoC28Rha and co-expressed in Nicotiana benthamiana. After the leaves were harvested, the leaves were extracted and analyzed by HPLC / MS in negative ionization mode. Among them, the leaf extract expressing only the genes required for the production of QA-TriF was used as a negative control. Peak 9 was detected at m / z=1247.5 in the leaf extract that only additionally expressed SoC28Rha, which is the expected [MH] of QA-TriFR. - ( Fig.10 ). In addition, the MS / MS fragmentation of peak 9 showed that the main fragment ion was: m / z = 955.4, which was consistent with [MH] - corresponding to; and m / z = 485.3, which is similar to [MH] - This indicates that after the C28 sugar chain breaks, the C3 sugar chain breaks ( Fig.10 ). Based on these results, it is inferred that Fig.10 Peak 9 in the figure is QA-TriFR, and SoC28Rha is rhamnosyltransferase.

[0417] Subsequently, the two enzymes SoC28Xyl1 and SoC28Xyl2 were characterized. When testing SoC28Xyl1, the genes required for the production of QA-TriFR (tHMGR+SobAS+SoC28C16+SoC23+SoCSL+SoC3Gal+SoC3Xyl+SoFuSyn+SoC28Fu+SoC28Rha) were co-expressed with candidate SoC28Xyl1 in Nicotiana benthamiana, and leaf extracts expressing only the genes required for the production of QA-TriFR were used as negative controls. Among them, peak 10 was detected in the sample expressing only SoC28Rha, the gene required for the production of QA-TriFR, with an m / z of 1379.6, which is the expected [MH] for QA-TriFR. - ( Fig.11 ). MS / MS results showed that the main fragment ions of peak 10 were m / z = 955.4 and m / z = 485.3. This indicates that the C28 sugar chain was first broken to form QA-Tri, and then the C3 sugar chain was broken to form saponin ( Fig.11). The activity assay method of SoC28Xyl2 was similar to that of SoC28Xyl1. The genes required for the production of QA-TriFRX (tHMGR+SobAS+SoC28C16+SoC23+SoCSL+SoC3Gal+SoC3Xyl+SoFuSyn+SoC28Fu+SoC28Rha+SoC28Xyl1) were co-expressed with candidate SoC28Xyl2 in Nicotiana benthamiana, and tobacco leaves expressing the genes required for the production of QA-TriFRX without the additional addition of candidate SoC28Xyl2 were used as negative controls. HPLC / MS analysis showed that the sample expressing only SoC28Xyl2 produced the expected [MH] at m / z=1511.6. - Peak 11( Fig.12 ). In addition, MS / MS analysis showed that the main fragment ions of peak 11 were [MHX] at m / z = 1379.6 and [MH-FRXX] at m / z = 955.4. - , and chlorophyll acid [MH] at m / z = 485.3 - ( Fig.12 ). Taken together, these results indicate that SoC28Xyl1 and SoC28Xyl2 are xylosyltransferases in Saponaria officinalis.

[0418] The above are the genes and enzymes required for the biosynthesis of QA-TriFRXX (11). The remaining steps of the saponin B biosynthesis pathway, namely the steps responsible for the transfer of 4-O-acetylquinovose to 13, are further described below. Although glycosyltransferases related to the biosynthesis of plant natural products generally belong to family 1 within the glycosyltransferase superfamily, no UGT in the above list of major candidate genes showed quinovose transferase activity against 11. Therefore, we further turned our attention to genes that were highly co-expressed with SobAS1 to expand the scope of the candidate gene search, and noticed that the candidate genes of glycosyl hydrolase family 1 (GH1) showed a high co-expression level with SobAS1 (PCC = 0.971) ( Figure 3 When SoGH1 was co-expressed with the biosynthetic gene of 11, two new products (12' and 12") were detected. These two products had different RTs but masses corresponding to the expected mass of 11+ deoxyhexose ([MH] - m / z=1657.7) is the same as ( Fig.13 In order to distinguish the two products, MS / MS analysis was performed on 12 and 12'. The results showed that their fragmentation patterns were the same, with the main fragment ion being m / z = 1525.7 ([MH] of QA-TriFRXX) -) and m / z = 955.4 ([MH] of QA-Tri) - ). This indicates that after the deoxyhexose is cleaved, the entire C-28 sugar chain is cleaved to generate QA-Tri. Subsequently, 12 and 12' were compared with the authentic standard 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponic acid (12, hereinafter abbreviated as QA-TriF(Q)RXX) and it was found that although the fragmentation conditions of 12 and 12' were consistent with those of QA-TriF(Q)RXX, only the RT of 12 was the same as that of the QA-TriF(Q)RXX standard. Based on these results, SoGH1 may be involved in the transfer of D-quinovose to QA-TriFRXX. After successfully elucidating the pathway of 12, only the acetylation step in the SpB(13) biosynthesis pathway remains to be elucidated. Figure 3 The list of leading candidate genes was screened for BAHD acetyltransferase function. LC / MS analysis of the resulting leaf extracts showed that co-expression of SoBAHD1 with the genome of generation 12 resulted in the formation of two basal acetyltransferases with the expected mass corresponding to SpB ([MH] - m / z = 1699.7) new products (13 and 13'). In addition, MS / MS analysis showed that 13 and 13' had the same fragmentation pattern, with the main fragment ion being m / z = 1657.7 ([MH] - ) and m / z = 955.5 (7 [M] - This indicates that after the acetyl group is broken, the entire C-28 sugar chain is broken ( Fig.14 ). However, only 13 produced by heterologous expression of SoBAHD1 corresponded to the SpB standard authentic in terms of RT and fragmentation pattern. Based on the above results, 13 was identified as SpB (13) produced after SoBAHD1 acetylated D-quinovose in 12, and SoBAHD1 was identified as an acetyltransferase capable of generating SpB by transferring the acetyl moiety to QA-TriF(Q)RXX.

[0419] The amino acid sequences (Table 6) were used to compare the sequence similarity of the saponin biosynthesis genes identified in this article with the corresponding genes involved in the biosynthesis of QS-21 in Quillaja chilensis. Although several genes at the beginning showed high similarity in amino acid sequence, the sequence similarity of the remaining pathway genes was generally low. This suggests that the two pathways may be independent pathways, providing evidence for convergent evolution. Fig.15 Shown is the saponin biosynthetic pathway described herein. However, the actual biosynthetic sequence in plants may be in any order.

[0420] In order to study the role of the genes characterized above in plants, hairy roots were successfully cultivated from Sodix flower seedlings. For proof of concept purposes, after silencing SobAS1 expression in Sodix flower hairy roots, the metabolic status of SobAS1-silenced hairy roots was compared with that of DsRED-expressing control hairy roots. Although β-amyrin was not detected in both the control hairy roots and the silenced hairy roots, Fig.16 ), but accumulation of cycloartenol was only observed in the SobAS1-silenced type ( Fig.17 ). This may indicate that silencing of SobAS1 in hairy roots of Sophora flavescens leads to increased flux to the sterol biosynthesis pathway. Further LC / MS analysis showed that SobAS1-silenced hairy roots did not accumulate quillabilis acid, while a large amount of quillabilis acid was detected in the control hairy roots ( Fig.18 ). Consistent with this result, SpB was not detected in SobAS1-silenced hairy roots, but was detected in control roots ( Fig.19 Taken together, these results indicate that SobAS1 is indeed the OSC responsible for the biosynthesis of saponin in saponaria.

[0421] sequence

[0422] SEQ ID NO: 1 Nucleotide sequence of SoC28 oxidase (SoC28)

[0423] MELFFICGLVLFSTLSLISLFLLHNHSSARGYRLPPGRMGWPFIGESYEFLANGWKGYPEKFIFSRLAKYKPNQVFKTSILGEKVAVMCGATCNKFLFSNEGKLVNAWWPNSVNKIFPSSTQTSSKEEAKKMRKLLPTFFKPEALQRYIPIMDEIAIRHMEDEWEGKSKIEVFPLAKRYTFWLACRLFLSIDDPVHVAKFADPFNDIASGIISIPIDLPGTPFNRGIKASNVVRQELKTIIKQRKLDLSDNKASPTQDILSHMLLTPDEDGRYMNELDIADKILGLLIGGHDTASAACTFVVKFLAELPHIYDGVYKEQMEIAKSKKEGERLNWEDIQKMKYSWNVACEVMRLAPPLQGAFREALSDFMYAGFQIPKGWKLYWSANSTHRNPECFPEPEKFDPARFDGSGPAPYTYVPFGGGPRMCPGKEYARLEILVFMHNIVKRFKWEKLIPDETIVVNPMPTPAKGLPVRLRPHSKPVTVSA*

[0424] SEQ ID NO: 2 Amino acid sequence of SoC28 oxidase (SoC28)

[0425]

[0426] SEQ ID NO: Nucleotide sequence of 3C28C16 oxidase (SoC28C16)

[0427] MELITLLSALLVLAIVSLSTFFVLYYNTPTKDGKTLPPGRMGWPFIGESYDFFAAGWKGKPESFIFDRLKKFAKGNLNGQFRTSLFGNKSIVVAGAAANKLLFSNEKKLVTMWWPPSIDKAFPSTAQLSANEEALLMRKFFPSFLIRREALQRYIPIMDDCTRRHFATGAWGPSDKIEAFNVTQDYTFWVACRVFMSIDAQEDPETVDSLFRHFNVLKAGIYSMHIDLPWTNFHHAMKASHAIRSAVEQIAKKRRAELAEGKAFPTQDMLSYMLETPITSAEDSKDGKAKYLNDADIGTKILGLLVGGHDTSSTVIAFFFKFMAENPHVYEAIYKEQMEVAATKAPGELLNWDDLQKMKYSWCAICEVMRLTPPVQGAFRQAITDFTHNGYLIPKGWKIYWSTHSTHRNPEIFPQPEKFDPTRFEGNGPPAFSFVPFGGGPRMCPGKEYARLQVLTFVHHIVTKFKWEQILPNEKIIVSPMPYPEKNLPLRMIARSESATLA*

[0428] SEQ ID NO: Amino acid sequence of 4C28C16 oxidase (SoC28C16)

[0429]

[0430] SEQ ID NO: 5 Nucleotide sequence of SoC23 oxidase (SoC23)

[0431] MEYLPYIATSIACIVILRWALNMMQWLWFEPRRLEKLLRKQGLQGNSYKFLFGDMKESSMLRNEALAKPMPMPFDNDYFPRINPFVDQLLNKYGMNCFLWMGPVPAIQIGEPELVREAFNRMHEFQKPKTNPLSALLATGLVSYEGDKWAKHRRLINPSFHVEKLKLMIPAFRESIVEVVNQWEKKVPENGSAEIDVWPSLTSLTGDVISRAAFGSVYGDGRRIFELLAVQKELVLSLLKFSYIPGYTYLPTEGNKKMKAVNNEIQRLLENVIQNRKKAMEAGEAAKDDLLGLLMDSNYKESMLEGGGKNKKLIMSFQDLIDECKLFFLAGHETTAVLLVWTLILLCKHQDWQTKAREEVLATFGMSEPTDYDALNRLKIVTMILNEVLRLYPPVVSTNRKLFKGETKLGNLVIPPGVGISLLTIQANRDPKVWGEDASEFRPDRFAEGLVKATKGNVAFFPFGWGPRICIGQNFALTESKMAVAMILQRFTFDLSPSYTHAPSGLITLNPQYGAPLMFRRR*

[0432] SEQ ID NO: 6 Amino acid sequence of SoC23 oxidase (SoC23)

[0433]

[0434] SEQ ID NO: 7 Nucleotide sequence of bAS (SobAS)

[0435] MWRLKIAEGGNDPYLYSTNNFVGRQTWEFDSEYGTPEAIKEVEEARQIFYKNRFQVKPCGDLLWRFQFLREKNFKQTIPQVKVGDGEEVTYEAASTTLKRSVNLLTALQADDGHWPAEIAGPQFFLPPLVFCLYITGHLNVVFNVHHREEILRSIYYHQNEDGGWGLHIEGHSTMFCTALNYICLRMLGVGPDEGDDNACPRARKWILDHGSVTHIPSWGKTWLSILGLFDWSGSNPMPPEFWILPTFMPMYPAKMWCYCRMVYMPMSYLYGKRFVGPITPLIKQLREELFSEPFEEIKWKKVRHLCAPEDLYYPHPLIQDLMWDSLYLFTEPLLTRWPFNNLIRQKALQVTMDHIHYEDENSRYITIGCVEKVLCMLACWVEDPNGVCYKKHLARVPDYIWIAEDGLKMQSFGSQQWDCGFAVQALLASNMSLDEIGPALKKGHFFIKESQVKDNPSGDFKSMHRHISKGSWTFSDQDHGWQVSDCTAEGLKCCLILSTMPPEIVGEKMDPERLYDSVNVLLSLQSENGGLSAWEPAGAQAWLELLNPTEFFADIVIEHEYVECTGASIQALVLFKKMYPGHRKKEIENFIAKAAKYLEDTQYPNGSWYGNWGVCFTYGTWFALGGLAAAGKTYANCAAMRKGVEFLLKSQKEDGGWGESYVSCPKKDFVPLEGPSNLTQTAWALMGLIYARQMERDPTPLHQAAKLLINSQLENGDFPQQEITGVFMKNCMLHYPMYRTIYPLWAIAEYRTHVPLRLS*

[0436] SEQ ID NO: 8 Amino acid sequence of bAS (SobAS)

[0437]

[0438] SEQ ID NO: 9 Nucleotide sequence of SoQA-GlcAT (SoCSL)

[0439] MSPHNTCTLQITRALLSRLHILFHSALVASVFYYRFSNFSSGPAWALMTFAELTLAFIWALTQAFRWRPVVRAVFGPEEIDPAQLPGLDVFICTADPRKEPVMEVMNSVVSALALDYPAEKLAVYLSDDGGSPLTREVIREAAVFGKYWVGFCGKYNVKTRCPEAYFSSFCDGERVDHNQDYLNDELSVKSKFEAFKKYVQKASEDATKCIVVNDRPSCVEIIHDSKQNGEGEVKMPLLVYVAREKRPGFNHHAKAGAINTLLRVSGLLSNSPFFLVLDCDMYCNDPTSARQAMCFHLDPKLAPSLAFVQYPQIFYNTSKNDIYDGQARAAFKTKYQGMDGLRGPVMSGTGYFLKRKALYGKPHDQDELLREQPTKAFGSSKIFIASLGENTCVALKGLSKDELLQETQKLAACTYESNTLWGSEVGYSYDCLLESTYCGYLLHCKGWISVYLYPKKPCFLGCATVDMNDAMLQIMKWTSGLIGVGISKFSPFTYAMSRISIMQSLCYAYFAFSGLFAVFFLIYGVVLPYSLLQGVPLFPKAGDPWLLAFAGVFISSLLQHLYEVLSSGETVKAWWNEQRIWIIKSITACLFGLLDAMLNKIGVLKASFRLTNKAVDKQKLDKYEKGRFDFQGAQMFMVPLMILVVFNLVSFFGGLRRTVIHKNYEDMFAQLFLSLFILALSYPIMEEIVRKARKGRS*

[0440] SEQ ID NO: 10 Amino acid sequence of SoQA-GlcAT (SoCSL)

[0441]

[0442] SEQ ID NO

[0443] MGSNTEATEIPKMPLKIVFLTLPIAGHMLHIVDTASTFAIHGVECTIITTPANVPFIEKSISATNTTIRQFLSIRLVDFHEAVGLPPGVENFSAVTCPDMRPKISKGLSIIQKPTEDLIKEISPDCIVSDMFYPWTSDFALEIGVPRVVFRGCGMFPMCCWHSIKSHLPHEKVDRDDEMIVLPTLPDHIEMRKSTLPDWVRKPTGYSYLMKMIDAAELKSYGVIVNSFSDLERDYEEYFKNVTGLKVWTVGPISLHVGRNEELEGSDEWVKWLDGKKLDSVIYVSFGGVAKFPPHQLREIAAGLESSGHDFVWVVRASDENGDQAEADEWSLQKFKEKMKKTNHGLVIESWVPQLMFLEHKAIGGMLTHVGWGTMLEGITAGLPLVTWPLYAEQFYNERLVVDVLKIGVGVGVKEFCGLDDIGKKETIGRENIEASVRLVMGDGEEAAAMRLLRVKELSEASMKAVREGGSSKANIHDFLNELSTLRSLRQA*

[0444] SEQ ID NO: 12QA-GalT(SoC3Gal) amino acid sequence

[0445]

[0446] SEQ ID NO: 13 Nucleotide sequence of SoQA-R XylT (SoC3Xyl)

[0447] MKSPLKLYFLPYISPGHMIPLSEMARLFANQGHHVTIITTTSNATLLQKYTTATLSLHLIPLPTKEAGLPDGLENFISVNDLETAGKLYYALSLLQPVIEEFITSNPPDCIVSDMFYPWTADLASQLQVPRMVFHAACIFAMCMKESMRGPDAPHLKVSSDYELFEVKGLPDPVFMTRAQLPDYVRTPNGYTQLMEMWREAEKKSYGVMVNNFYELDPAYTEHYSKIMGHKVWNIGPAAQILHRGSGDKIERVHKAVVGENQCLSWLDTKEPNSVFYVCFGSAIRFPDDQLYEIASALESSGAQFIWAVLGKDSDNSDSNSDSEWLPAGFEEKMKETGRGMIIRGWAPQVLILDHPSVGGFMTHCGWNSTIEGVSAGVGMVTWPLYAEQFYNEKLITQVLKIGVEAGVEEWNLWVDVGRKLVKREKIEAAIRAVMGEAGVEMRRKAKELSVKAKKAVQDGGSSHRNLMALIEDLQRIRDDKMSKVAN*

[0448] SEQ ID NO: 14 Amino acid sequence of SoQA-R XylT (SoC3Xyl)

[0449]

[0450] SEQ ID NO: 15 Nucleotide sequence of QATriFuT(SoC28F)

[0451] MSDQNDKKVEIIVFPYHGQGHMNTMLQFAKRIAWKNAKVTIATTLSTTNKMKSKVENAWGTSITLDSIYDDSDESQIKFMDRMARFEAAAASSLSKLLVQKKEEADNKVLLVYDGNLPWALDIAHEHGVRGAAFFPQSCATVATYYSLYQETQGKELETELPAVFPPLELIQRNVPNVFGLKFPEAVVAKNGKEYSPFVLFVLRQCINLEKADLLLFNQFDKLVEPGEVLQWMSKIFNVKTIGPTLPSSYIDKRIKDDVDYGFHAFNLDNNSCINWLNSKPARSVIYIAFGSSVHYSVEQMTEIAEALKSQPNNFLWAVRETEQKKLPEDFVQQTSEKGLMLSWCPQLDVLVHESISCFVTHCGWNSITEALSFGVPMLSVPQFLDQPVDAHFVEQVWGAGITVKRSEDGLVTRDEIVRCLEVLNNGEKAEEIKANVARWKVLAKEALDEGGSSDKHIDEIIEWVSSF*

[0452] SEQ ID NO: 16 Amino acid sequence of QATriFuT(SoC28F)

[0453]

[0454] SEQ ID NO: 17 Nucleotide sequence of QA-TriFR(SoC28Rha)

[0455] MSAKMLHVVMYPWFAYGHMIPFLHLSNKLAETGHKVTYILPPKALTRLQNLNLNPTQITFRTITVPRVDGLPAGAENVTDIPDITLHTHLATALDRTRPEFETIVELIKPDVIMYDVAYWVPEVAVKYGAKSVAYSVVSAASVSLSKTVVDRMTPLEKPMTEEERKKKFAQYPHLIQLYGPFGEGITMYDRLTGMLSKCDAIACRTCREIEGKYCQYLSTQYEKKVTLTGPVLPEPEVGATLEAPWSEWLSRFKLGSVLFCAFGSQFYLDKDQFQEIILGLEMTNLPFLMAVQPPKGCATIEEAYPEGFAERVKDRGVVTSQWVQQLVILAHPAVGCFVNHCAFGTMWEALLSEKQLVMIPQLGDQILNTKMLADELKVGVEVERGIGGWVSKENLCKAIKSVMDEDSEIGKDVKQSHEKWRATLSSKDLMSTYIDSFIKDLQALVE*

[0456] SEQ ID NO: 18 Amino acid sequence of QA-TriFR(SoC28Rha)

[0457]

[0458] SEQ ID NO: 19 Nucleotide sequence of SoQA-TriFRXylT (SoC28Xyl1)

[0459] MGTKELHIVMYPWLAFGHFIPYLHLSNKLAQKGHKITFLLPHRAKLQLDSQNLYPSLITLVPITVPQVDTLPLGAESTADIPLSQHGDLSIAMDRTRPEIESILSKLDPKPDLIFFDMAQWVPVIASKLGIKSVSYNIVCAISLDLVRDWYKKDDGSNVPSWTLKHDKSSHFGENISILERALIALGTPDAIGIRSCREIEGEYCDSIAERFKKPVLLSGTTLPEPSDDPLDPKWVKWLGKFEEGSVIFCCLGSQHVLDKPQLQELALGLEMTGLPFFLAIKPPLGYATLDEVLPEGFSERVRDRGVAHGGWVQQPQMLAHPSVGCFLCHCGSSSMWEALVSDTQLVLFPQIPDQALNAVLMADKLKVGVKVEREDDGGVSKEVWSRAIKSVMDKESEIAAEVKKNHTKWRDMLINEEFVNGYIDSFIKDLQDLVEK*

[0460] SEQ ID NO: 20 Amino acid sequence of SoQA-TriFRXylT (SoC28Xyl1)

[0461]

[0462] SEQ ID NO: 21 SoQA-TriFRXXylT (SoC28Xyl2) nucleotide sequence

[0463] MEESKEEVHVAFFPFMTPGHSIPMLDLVRLFIARGVKTTVFTTPLNAPNISKYLNIIQDSSSNKNTIYVTPFPSKEAGLPEGVESQDSTTSPEMTLKFFVAMELLQDPLDVFLKETKPHCLV ADNFFPYATDIASKYGIPRFVFQFTGFFPMSVMMALNRFHPQNSVSSDDDPFLVPSLPHDIKLTKSQLQREYEGSDGIDTALSRLCNGAGRALFTSYGVIFNSFYQLEPDYVDYYTNTMGKRS RVWHVGPVSLCNRRHVEGKSGRGRSASISEHLCLEWLNAKEPNSVIYVCFGSLTCFSNEQLKEIATALERCEEYFIWVLKGGKDNEQEWLPQGFEERVEGKGLIIRGWAPQVLILDHEAIGGF VTHCGWNSTLESISAGVPMVTWPIYAEQFYNEKLVTDVLKVGVKVGSMKWSETTGATHLKHEEIEKALKQIMVGEEVLEMRKRASKLKEMAYNAVEEGGSSYSHLTSLIDDLMASKAVLQKF*

[0464] SEQ ID NO: 22SoQA-TriFRXXylT (SoC28Xyl2) amino acid sequence

[0465] The full-length HMGR sequence is shown below. The 5' region (underlined) can be removed to form a truncated, non-feedback-sensitive form (tHMGR). In addition, the sequence of tHMGR is also given below.

[0466] ATGGCTGTGGAGGTTCACCGCCGGGCTCCCGCGCCCCATGGCCGGGGCACCGGGGAGAAGGGCCGCGTG CAGGCCGGGGACGCGCTGCCGCTGCCGATCCGCCACACCAACCTCATCTTCTCGGCGCTCTTCGCCCGCCTCCCTCGC ATACCTCATGCGCCGCTGGAGGGAGAAGATCCGCAACTCCACGCCGCTCCACGTCGTGGGGCTCACCGAGATCTTCG CCATCTGCGGCCTCGTCGCCTCCCTCATCTACCTCCTCAGCTTCTTCGGCATCGGCTTTCGTGCAGTCCGTCGTATCC AACAGCGACGACGAGGACGAGGACTTCCTCATCGCGGCTGCAGCATCCCAGGCCCCCCCGCCGCCCTCCTCCAAGCC CGCGCCGCAGCAGTGCGCCCTGCTGCAGAGCGCCGGAGTC

[0467] SEQ ID NO: 23 AsHMGR (Avena strigosa HMG-CoA reductase) coding sequence (1689 bp)

[0468] MAVEVHRRAPAPHGRGTGEKGRVQAGDALPLPIRHTNLIFSALFAASLAYLMRRWREKIRNSTPLHVV GLTEIFAICGLVASLIYLLSFFGIAFVQSVVSNSDDEDDEDFLIAAAASQAPPPPSSKPAPQQCALLQSAGV APEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDGFDYDSILGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCLIASTNRGCKAIAESGGASSVVYRDGMTRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFTCSTGDAMGMNMVSKGVQNVLD YLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSVVCEAVIREEVVHKVLKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVESS QCITMLEAVNDGRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVLAGELSLISAQAAGHLVQSHMKYNRSSKDMSKIAC*

[0469] SEQ ID NO: 24 AsHMGR (Avena strigosa HMG-CoA reductase) translation nucleotide sequence (562aa)

[0470]

[0471] SEQ ID NO: 25 AstHMGR (Avena strigosa truncated HMG-CoA reductase) coding sequence (1275 bp)

[0472] MAPEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDGFDYDSILGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCLIASTNRGCKAIAESGGASSVVYRDGMTRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFTCSTGDAMGMNMVSKGVQNVL DYLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSVVCEAVIREEVVHKVLKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVES SQCITMLEAVNDGRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVLAGELSLISAQAAGHLVQSHMKYNRSSKDMSKIAC*

[0473] SEQ ID NO: 26 AstHMGR (Avena strigosa truncated HMG-CoA reductase) translated nucleotide sequence (424aa)

[0474]

[0475] SEQ ID NO: 27 AsSQS (Avena strigosa squalene synthase) coding sequence (1212 bp)

[0476] MGALSRPEEVVALVKLRVAAGQIKRQIPAEEHWAFAYDMLQKVSRSFALVIQQLGPELRNAVCIFYLVLRALDTVEDDTSIPNDVKLPILRDFYRHVYNPD WRYSCGTNHYKVLMDKFRLVSTAFLELGEGYQKAIEEITRRMGAGMAKFICQEVETIDDYNEYCHYVAGLVGYGLSRLFHAAGTEDLASDQLSNSMGLFLQ KTNIIRDYLEDINEIPKCRMFWPREIWSKYADKLEDLKYEENSEKAVQCLNDMVTNALVHAEDCLQYMSALKDNTNFRFCAIPQIMAIGTCAICYNNVKVFRGVVKMRRGLTARIIDETKSMSDVYSAFYEFSSLLESKIDDNDPSSALTRKRVEAIKRTCKSSGLLKRRGYDLEKSKYRHMLIMLALLLVAIIFGVLYAK*

[0477] SEQ ID NO: 28 AsSQS (Avena strigosa squalene synthase) translated nucleotide sequence (403aa)

[0478]

[0479] SEQ ID NO: 29 Coding sequence (2325 bp) of AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2)

[0480] MKNMMNYKLKLCSVSKNSKGVSLSPTPHLTKPPTIHTERDLLLPSSSFFFLLLSSSSYNIYNAMSSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTSIAVLIGCIVMLVWRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETGDHVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFPPCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAVPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFFGCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW*

[0481] SEQ ID NO: 30 Translated nucleotide sequence (774 aa) of AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2)

[0482] ATGGCTGAAGCATCCTCATTTCTTGCACAGAAAAGGTATGCGGTCGTGACAGGAGCAAACAAAGGACTAGGACTAGAAATATGCGGACAGCTTGCTTCACAGGGGGTGACGGTACTGCTGACATCCAGAGATGAAAAACGAGGCTTAGAAGCCATTGAGGAGCTTAAGAAATCGGGGATTAATTCGGAAAATCTTGAATATCATCAGCTGGATGTTACTAAGCCAGCTAGTTTCGCTTCTCTGGCCGATTTCATCAAGGCCAAATTTGGCAAGCTTGATATCCTGGTGAACAATGCAGGGATCAGCGGTGTTATTGTAGATTATGCAGCTTTAATGGAAGCCATTCGCCGTCGAGGGGCAGAGATCAATTACGATGGAGTGATGAAACAGACCTACGAGCTAGCAGAGGAATGCTTGCAAACAAATTACTATGGTGTGAAAAGAACCATTAATGCTCTCCTTCCGCTACTTCAGTTTTCCGATTCACCAAGGATCGTCAATGTTTCCTCCGATGTTGGCCTCCTTAAGAAAATACCCGGCGAGAGAATCAGAGAAGCCTTAGGCGACGTGGAAAAACTTACGGAAGAAAGCGTGGACGGGATTTTAGACGAGTTTCTAAGAGATTTCAAGGAAGGCAAGATCGCAGAGAAAGGTTGGCCTACGTTTAAGAGCGCCTATTCAATCTCAAAGGCGGCGCTCAATTCGTACACGAGGGTTTTAGCACGGAAATACCCGTCGATCATCATCAACTGTGTCTGCCCGGGTGTCGTCAAAACCGATATCAATCTTAAAATGGGCCACTTGACGGTTGAAGAAGGCGCGGCCAGTCCCGTGAGGTTAGCACTCATGCCCCTTGGTTCGCCTTCCGGCCTGTTCTATACTCGAAACGAAGTAACTCCATTTGAATGA

[0483] SEQ ID NO: 31 SoFuSyn coding sequence

[0484] MAEASSFLAQKRYAVVTGANKGLGLEICGQLASQGVTVLLTSRDEKRGLEAIEELKKSGINSENLEYHQLDVTKPASFASLADFIKAKFGKLDILVNNAGISGVIVDYAALMEAIRRRGAEINYDGVMKQTYELAEECLQTNYYGVKRTINALLPLLQFSDSPRIVNVSSDVGLLKKIPGERIREALGDVEKLTEESVDGILDEFLRDFKEGKIAEKGWPTFKSAYSISKAALNSYTRVLARKYPSIIINCVCPGVVKTDINLKMGHLTVEEGAASPVRLALMPLGSPSGLFYTRNEVTPFE*

[0485] SEQ ID NO: 32 Nucleotide sequence translated by SoFuSyn

[0486]

[0487] SEQ ID NO: 33 Coding sequence of SoGH1

[0488] MVLSRLDFPSDFIFGSGTSASQVEGAALEDGKTSTAFEGFLTRMSGNDLSKGVEGYYKYKEDVQLMVQTGLDAYRFSISWSRLIPGGKGPVNPKGLQYYNNFIDELIKNGIQPHVTLLHFDIPDTLMTAYNGLKGQEFVEDFTAFADVCFKEFGDRVLYWTTVNEANNFASLTLDEGNFMPSTEPYIRGHNIILAHASAVKLYREKYKKTQNGFIGLNLYASWYFPETDDEQDSIAAQRAIDFTIGWIMQPLIYGEYPETLKKQVGERLPTFTKEESTFVKNSFDFIGVNCYVGTAVKDDPDSCNSKNKTIITDMSAKLSPKGELGGAYMKGLLEYFKRDYGNPPIYIQENGYWTPRELGVNDASRIEYHTASLASMHDAMKNGA

[0489] SEQ ID NO: 34 Translated nucleotide sequence of SoGH1

[0490]

[0491] SEQ ID NO: 35 SoBAHD1 coding sequence

[0492] MEPSKMEVKIISSETIKPSSPTPSHLRKYTLSLLDQKYTPIVVPAILFYERPQGVAPLDMDRLRTCLSQTLTAFYPLAGRAESRDVIICNDEGIPFVEAHVDCELSSVVKSLSS LGSDLRSFYPPRDGLLEGGIQFAIQMNVFSCGGFAFAWYCTHNVTDGTSTANFFRYWTALYAQRSEYAVQDLMDFNSVVTAFPPVPPRVPQEEKPVTTELKPEKQEGQEKEEKKK SSFNFSFQSHIVARSFLIKSKAVAELKAKSVSEEVPYPSRFEAVSAFLWKSIVSSSSTTEGKTMINMPVNLRPRVDPPLPLDSVGNIFENALVQSEKKAELHEFVARIRGSISKMK DFATEYQGEKREEAKDAHWKRFIKAVIECKGKDAYVISPWYKSSGFTDIDFGFGTPIRVVPMDDVVNHNQRNTIMLMEFVDSDGDGFEAWMFLEEECIKFLESNPEFLAFASPNF

[0493] SEQ ID NO: 36 SoBAHD1 translated nucleotide sequence

[0494] surface

[0495]

[0496] Table 1 List of published squalene cyclase sequences used in phylogenetic analysis

[0497]

[0498] Table 2 Primer oligonucleotide sequences

[0499]

[0500] Table 3 Correlation analysis between candidate CYP450 and characterized SobAS gene expression patterns using Pearson correlation coefficient (PCC)

[0501]

[0502] Table 4 Correlation analysis between candidate UGTs and characterized SobAS gene expression patterns using Pearson correlation coefficient (PCC)

[0503]

[0504] Table 5 Correlation analysis between candidate CSLs and characterized SobAS gene expression patterns using Pearson correlation coefficient (PCC)

[0505]

[0506] Table 6 Amino acid sequence similarity between genes involved in saponin biosynthesis in Soapwort and QS-21 biosynthesis genes in Quillaja chinensis

[0507] References

[0508] [1] Jia Z, Koike K, Nikaido T. Major triterpenoid saponins from Saponaria officinalis. Journal of Natural Products, 61, 1368–1373, 1998.

[0509] [2] Eastman, J., Wildflowers of the Eastern United States: An Introduction to Common Species of Woods, Wetlands and Fields, Stackpole Books, 2014.

[0510] [3] Rees, A., The cyclopcedia; or, universal dictionary of arts, sciences, and literature (Volume 4), Longman, Hurst, Rees, Orme and Brown, 1819.

[0511] [4] Korkmaz M., Ozelik Economic significance of plants of the genera Gypsophila L., Ankyropetalum fenzl. and Saponaria L. (Caryophyllaceae), African Journal of Biotechnology, Vol. 10, No. 47, pp. 9533-9541.

[0512] [5] Porter Melzig, M.F. Triterpenoid saponins from Caryophyllaceae and Illecebraceae. Phytochemistry Letters, 4, 2011, 59–68.

[0513] [6] Smulek Zdarta A, Pacholak A, Zgragrzeskowiak A. Markzak Yaribski Kaczorek E., Saponaria extracts: surface activity and effects on environmental strains, Colloids and Surfaces B: Biointerfaces, 2017, 150, 209-215.

[0514] [7] Gonzalez P.J., Sorensen Characterization of soapwort saponin foam for food applications, Food Hydrocolloids, 2020, 101, 105541.

[0515] [8] Sadowska B, Budzyńska A, Wijkovska Sakir -Szakiel M, Paszkiewicz M, Stochmal A, Moniuszko-Szajwaj B, Kowalczyk M, Rozalska New pharmacological properties of saponin-rich fractions of Medicago sativa and Soapwort against Candida albicans, Journal of medical microbiology, 2014, Vol. 63, No. 8, pp. 1076-1086.

[0516] [9] Gilabert-Oriol R, Thakur M, Haussmann K, Niesler N, Bhargava C, Golik Fuchs H, Weng A. Potentiation of rituximab immunotoxin by saponins from the genus Saponaria. Planta Medicina, 2016, Vol. 82, No. 18, pp. 1525-1531.

[0517]

[10] Reed J, Oum A, Eldmerdash A, Owen C, Martin L B, Misra R C, …, Osborne A, Evaluation of the biosynthetic pathway of the Chilean Quillaja saponin adjuvant, Science, 2023, Vol. 379, No. 6638, pp. 1252–1264.

Claims

1. A method for producing triterpenes, comprising: (i) contacting 2,3-epoxysqualene with a Saponaria officinalis β-amyrin synthase (SobAS) comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8, so that the 2,3-epoxysqualene is converted into β-amyrin; (ii) any of the following: a) contacting β-amyrin with a SoC28 oxidase polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2, such that the C-28 position of the β-amyrin is oxidized to a carboxylic acid, thereby producing oleanolic acid, and contacting the oleanolic acid with a SoC28C16 oxidase polypeptide comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 4, so that the C-16 position of the oleanolic acid is oxidized to an alcohol, thereby producing echinocystic acid; or b) contacting oleanolic acid with a SoC28C16 oxidase polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 4, such that the C-16 position of the oleanolic acid is oxidized to an alcohol, and oxidizing the C-28 position of the β-amyrin to a carboxylic acid, thereby generating echinocystic acid; (iii) contacting the echinocystic acid with a SoC23 oxidase polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 6, such that the C-23 position of the echinocystic acid is oxidized to an aldehyde, thereby generating quince acid (QA); (iv) contacting QA with a Soapwort QA3-O-glucuronyltransferase (SoCSL) polypeptide comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10, such that QA is converted to QA-GlcA; (v) contacting QA-GlcA with a Soapwort QA-GlcA galactosyltransferase (SoC3Gal) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 12, such that the QA-GlcA is converted to QA-GlcA-Gal; (vi) contacting QA-GlcA with a Soapwort QA-GlcA-Gal xylosyltransferase (SoC3Xyl) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 14, such that QA-GlcA-Gal is converted to QA-Tri; (vii) contacting QA-Tri with a Soapwort QA-Tri fucosyltransferase (SoC28Fu) polypeptide comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 16, such that the QA-Tri is converted to QA-TriF; (viii) contacting QA-TriF with a Soapwort QA-TriF rhamnosyltransferase (SoC28Rha) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 18, such that the QA-TriF is converted to QA-TriFR; (ix) contacting QA-TriFR with a Soapwort QA-TriFR xylosyltransferase (SoC28Xyl1) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 20, such that the QA-TriFR is converted to QA-TriFRX; (x) contacting QA-TriFRX with a Soapwort QA-TriFRX xylosyltransferase (SoC28Xyl2) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 22, such that the QA-TriFRX is converted into QA-TriFRXX; (xi) contacting QA-TriFRXX with a Soapwort QA-TriFRXX quinoside transferase (SoGH1) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 34, such that QA-TriFRXX is converted to QA-TriF(Q)RXX; and / or (xii) contacting QA-TriF(Q)RXX with a Saponaria officinalis QA-TriF(Q)RXX acetyltransferase (SoBAHD1) polypeptide comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:36, such that QA-TriF(Q)RXX is converted to saponin B (SpB).

2. The method according to claim 1, comprising: (i) Any of the following: (a) contacting β-amyrin with a saponaria officinalis C28 oxidase (SoC28 oxidase) to oxidize the C-28 position of the β-amyrin to a carboxylic acid, thereby forming oleanolic acid, wherein the amino acid sequence of the SoC28 oxidase has at least 80% sequence identity with SEQ ID NO: 2; and contacting oleanolic acid with a saponaria officinalis C28C16 oxidase (SoC28C16 oxidase) to oxidize the C-16 position of the oleanolic acid to an alcohol, thereby forming echinocystic acid, wherein the amino acid sequence of the C16 oxidase has at least 50% sequence identity with SEQ ID NO: 4; or (b) contacting β-amyrin with a saponaria officinalis C28C16 oxidase (SoC28C16 oxidase) to oxidize the C-28 position of the β-amyrin to a carboxylic acid and the C-16 position thereof to an alcohol, thereby forming echinococcal acid, wherein the amino acid sequence of the C28C16 oxidase has at least 50% sequence identity with SEQ ID NO: 4; (iii) contacting echinocystic acid with a saponin C-23 oxidase (SoC23 oxidase) to oxidize the C-23 position of echinocystic acid to an aldehyde, thereby forming quince acid (QA), wherein the amino acid sequence of the SoC23 oxidase has at least 50% sequence identity with SEQ ID NO:

6.

3. The method according to claim 2, wherein: β-Amyrin is produced by contacting 2,3-epoxysqualene (OS) with a β-amyrin synthase (SobAS) having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8 to cyclize the 2,3-epoxysqualene to produce β-amyrin.

4. The method according to claim 2 or claim 3, further comprising: (iv) contacting QA with a Soapwort QA 3-O glucuronyltransferase (SoCSL) to covalently attach D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA), wherein the amino acid sequence of the SoCSL has at least 60% sequence identity to SEQ ID NO: 10; (v) contacting QA-GlcA with a Soapwort QA-GlcA galactosyltransferase (SoC3Gal) to covalently attach D-galactose (Gal) to QA-GlcA via a β-1→2 bond, thereby forming 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-soapwort acid (QA-GlcA-Gal), wherein the amino acid sequence of QA-GlcA-Gal has at least 50% sequence identity to SEQ ID NO: 12; and (vi) contacting QA-GlcA-Gal with a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) to covalently attach D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin ("QA-GlcA-[Gal]-Xyl" QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO:

14.

5. The method according to claim 4, further comprising: (vii) contacting 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-Tri) with a Saponaria officinalis QA-Tri fucosyltransferase (SoC28Fu) to attach fucose to the 28-O position of QA-Tri, thereby forming 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), wherein the amino acid sequence of QATriFuT has at least 60% sequence identity to SEQ ID NO: 16; (viii) contacting QA-TriF with a saponin QA-TriF rhamnosyltransferase (SoC28Rha) to covalently attach rhamnose to QA-TriF via a 1,2 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFR), wherein the amino acid sequence of SoC28Rha has at least 50% sequence identity to SEQ ID NO: 18; (ix) contacting QA-TriFR with a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) to covalently attach xylose to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRX), wherein the amino acid sequence of SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; and (x) contacting QA-TriFRX with a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) to covalently attach xylose to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 50% sequence identity to SEQ ID NO:

22.

6. The method according to claim 5, further comprising: contacting QA-TriFRXX with a saponin QA-TriFRXX quinovosyltransferase (SoGH1) to covalently attach quinovose to QA-TriFRXX via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and QA-TriF(Q)RXX is contacted with Soapberry QA-TriF(Q)RXX acetyltransferase (SoBAHD1) to covalently attach an acetyl group to QA-TriF(Q)RXX to form QA-TriF(Q-Ac)RXX (saponin B), wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity with SEQ ID NO:

36.

7. A method for converting a host from a phenotype in which the host is unable to perform triterpene biosynthesis from 2,3-epoxysqualene (OS) to a phenotype in which the host is capable of performing triterpene biosynthesis, the method comprising: expressing a heterologous nucleic acid in said host or one or more cells thereof, following a step of introducing said nucleic acid into said host or an ancestor of either host, Wherein, the heterologous nucleic acid encodes one or more of the following: (i) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid, wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2; (ii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and the C-16 position thereof to an alcohol (C16 oxidase) to form echinocostic acid, wherein the SoC28C16 oxidase has a sequence identity of at least 50% to SEQ ID NO: 4; (iii) a SoC23 oxidase capable of oxidizing the C-23 position of echinocystic acid to an aldehyde to form quince acid (QA), wherein the SoC23 oxidase has at least 50% sequence identity to SEQ ID NO: 6; (iv) a Soapwort QA 3-O glucuronyltransferase (SoCSL) that attaches D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA), wherein the SoCSL has a sequence identity of at least 60% to SEQ ID NO: 10; (v) a saponin QA-GlcA galactosyltransferase (SoC3Gal) that attaches D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-Gal), wherein the amino acid sequence of the SoC3Gal has at least 50% sequence identity to SEQ ID NO: 12; (vi) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) that attaches D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-[Gal]-Xyl, QA-Tri), wherein the amino acid sequence of the SoC3Xyl has at least 50% sequence identity to SEQ ID NO: 14; (vii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) that attaches fucose (Fuc) to the 28-O position of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), wherein SoC28Fu has a sequence identity of at least 60% to SEQ ID NO: 16; (viii) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) that attaches rhamnose (Rha) to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFR), wherein SoC28Rha has at least 50% sequence identity to SEQ ID NO: 18; (ix) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) that attaches D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of the SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; (x) a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 80% sequence identity to SEQ ID NO: 22; (xi) a saponin QA-TriFRXX quinovose transferase (SoGH1) that attaches quinovose to QA-TrFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and / or (xii) a Soapberry QA-TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to QA-TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO:

36.

8. The method according to claim 7, wherein: The heterologous nucleic acid encodes the following polypeptide: (i) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid, wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2; (ii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and the C-16 position thereof to an alcohol (C16 oxidase) to form echinocystic acid, wherein the SoC28C16 oxidase has a sequence identity of at least 50% to SEQ ID NO: 4; and (iii) a SoC23 oxidase capable of oxidizing the C-23 position of echinocystic acid to an aldehyde to form quisac acid (QA), wherein the SoC23 oxidase has a sequence identity of at least 50% to SEQ ID NO:

6.

9. The method according to claim 8, wherein: The heterologous nucleic acid further encodes a saponin beta-amyrin synthase (SobAS) that cyclizes 2,3-epoxysqualene to triterpenes, wherein the SobAS has at least 80% sequence identity to SEQ ID NO:

8.

10. The method according to claim 8 or claim 9, wherein: The heterologous nucleic acid further encodes the following polypeptide: (iv) a Soapwort QA3-O glucuronyltransferase (SoCSL) that attaches D-glucuronic acid (GlcA) to the 3-O position of saponic acid to form 3-O-{β-D-pyranoglucuronic acid}-saponic acid (QA-GlcA), wherein the SoCSL has a sequence identity of at least 60% to SEQ ID NO: 10; (v) a saponin QA-GlcA galactosyltransferase (SoC3Gal) that attaches D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-Gal), wherein the amino acid sequence of the SoC3Gal has at least 50% sequence identity to SEQ ID NO: 12; and (vi) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) that attaches D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal-Xyl or QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO:

14.

11. The method according to claim 10, wherein: The heterologous nucleic acid further encodes the following polypeptide: (vii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) that attaches fucose (Fuc) to the 28-O position of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), wherein SoC28Fu has a sequence identity of at least 60% to SEQ ID NO: 16; (viii) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) that attaches rhamnose (Rha) to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFR), wherein SoC28Rha has at least 50% sequence identity to SEQ ID NO: 18; (ix) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) that attaches D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of the SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; and (x) a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 80% sequence identity to SEQ ID NO:

22.

12. The method according to claim 11, wherein: The heterologous nucleic acid further encodes the following polypeptide: (xi) a saponin QA-TriFRXX quinovose transferase (SoGH1) that attaches quinovose (Q) to QA-TrFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and (xii) a Soapberry QA-TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to QA-TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO:

36.

13. A host cell comprising or transformed with a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a plurality of nucleotide sequences, each nucleotide sequence encoding a polypeptide, wherein the polypeptides in combination have triterpene biosynthetic activity, in, The multiple nucleotide sequences encode one or more of the following polypeptides: (i) a saponin beta-amyrin synthase (SobAS) that cyclizes 2,3-epoxysqualene to triterpenes, wherein the SobAS has a sequence identity of at least 80% to SEQ ID NO: 8; (ii) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid, wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2; (iii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and the C-16 position thereof to an alcohol (C16 oxidase) to form echinocystic acid, wherein the SoC28C16 oxidase has a sequence identity of at least 50% to SEQ ID NO: 4; (iv) a SoC23 oxidase capable of oxidizing the C-23 position of echinocystic acid to an aldehyde to form quisac acid (QA), wherein the SoC23 oxidase has at least 50% sequence identity to SEQ ID NO: 6; (v) a Soapwort sapote 3-O glucuronyltransferase (SoCSL) that attaches D-glucuronic acid (GlcA) to the 3-O position of sapote to form 3-O-{β-D-pyranoglucuronic acid}-sapote (QA-GlcA), wherein the SoQA-GlcT has a sequence identity of at least 60% to SEQ ID NO: 10; (vi) a saponin QA-GlcA galactosyltransferase (SoC3Gal) that attaches D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-Gal), wherein the amino acid sequence of the QA-GlcA-Gal has at least 50% sequence identity to SEQ ID NO: 12; (vii) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) that attaches D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-[Gal]-Xyl, QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO: 14; (viii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) that attaches fucose (Fuc) to the 28-O position of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), wherein the SoC28Fu has a sequence identity of at least 60% to SEQ ID NO: 16; (ix) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) that attaches rhamnose (Rha) to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-pyranofucoidsyl ester}-saponin acid (QA-TriFR), wherein SoC28Rha has at least 50% sequence identity to SEQ ID NO: 18; (x) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) that attaches D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of the SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; and / or (xi) a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 50% sequence identity to SEQ ID NO: 22; (xii) a saponin QA-TriFRXX quinovose transferase (SoGH1) wherein quinovose (Q) is attached to QA-TriFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and / or (xiii) a Soapwort QA-TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to QA-TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO: 36, Wherein, the expression of the nucleic acid enables the transformed host to have the ability to carry out triterpene biosynthesis.

14. The host cell according to claim 13, wherein The various nucleotide sequences encode the following polypeptides: (i) a SoC28 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid to form oleanolic acid, wherein the SoC28 oxidase has a sequence identity of at least 80% to SEQ ID NO: 2; (ii) a SoC28C16 oxidase capable of oxidizing the C-28 position of β-amyrin to a carboxylic acid and the C-16 position thereof to an alcohol (C16 oxidase) to form echinocystic acid, wherein the SoC28C16 oxidase has a sequence identity of at least 50% to SEQ ID NO: 4; and (iii) a SoC23 oxidase capable of oxidizing the C-23 position of echinocystic acid to an aldehyde to form quisac acid (QA), wherein the SoC23 oxidase has a sequence identity of at least 50% to SEQ ID NO: 6, The expression of the nucleic acid enables the transformed host to have the ability to carry out saponin biosynthesis.

15. The host cell according to claim 14, wherein The heterologous nucleic acid further encodes a saponin beta-amyrin synthase (SobAS) that cyclizes 2,3-epoxysqualene to triterpenes, wherein the SobAS has at least 80% sequence identity to SEQ ID NO:

8.

16. The host cell according to claim 14 or claim 15, wherein The heterologous nucleic acid further encodes the following polypeptide: (iv) a Soapwort sapote 3-O glucuronyltransferase (SoCSL) that attaches D-glucuronic acid (GlcA) to the 3-O position of sapote to form 3-O-{β-D-pyranoglucuronic acid}-sapote (QA-GlcA), wherein the SoCSL has a sequence identity of at least 60% to SEQ ID NO: 10; (v) a saponin QA-GlcA galactosyltransferase (SoC3Gal) that attaches D-galactose (Gal) to QA-GlcA via a β-1→2 bond to form 3-O-{[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin acid (QA-GlcA-Gal), wherein the amino acid sequence of the SoC3Gal has at least 50% sequence identity to SEQ ID NO: 12; and (vi) a saponin QA-GlcA-Gal xylosyltransferase (SoC3Xyl) that attaches D-xylose (Xyl) to QA-GlcA-Gal via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-saponin (QA-GlcA-Gal-Xyl, QA-Tri), wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity with SEQ ID NO:

14.

17. The host cell according to claim 16, wherein The heterologous nucleic acid further encodes one, two, three or all four of the following polypeptides: (vii) a Soapwort QA-Tri fucosyltransferase (SoC28Fu) that attaches fucose (Fuc) to position 28-O of QA-Tri to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin (QA-TriF), wherein SoC28Fu has a sequence identity of at least 60% to SEQ ID NO: 16; (viii) a saponin QA-TriF rhamnosyltransferase (SoC28Rha) that attaches rhamnose (Rha) to QA-TriF via a 1,2 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFR), wherein SoC28Rha has at least 50% sequence identity to SEQ ID NO: 18; (ix) a saponin QA-TriFR xylosyltransferase (SoC28Xyl1) that attaches D-xylose (Xyl) to QA-TriFR via a 1,4 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRX), wherein the amino acid sequence of the SoC28Xyl1 has at least 50% sequence identity to SEQ ID NO: 20; and (x) a saponin QA-TriFRX xylosyltransferase (SoC28Xyl2) that attaches D-xylose (Xyl) to QA-TriFRX via a 1,3 bond to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranosyl ester}-saponin acid (QA-TriFRXX), wherein the amino acid sequence of SoC28Xyl2 has at least 50% sequence identity to SEQ ID NO:

22.

18. The host cell according to claim 17, wherein The heterologous nucleic acid further encodes one or both of the following polypeptides: (xi) a saponin QA-TriFRXX quinovose transferase (SoGH1) that attaches quinovose (Q) to QA-TrFRXX via a 1,4 linkage to form 3-O-{β-D-xylopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranosyl ester}-saponin acid (QA-TriF(Q)RXX), wherein the amino acid sequence of SoGH1 has at least 50% sequence identity to SEQ ID NO: 34; and (xii) a Saponaria TriF(Q)RXX acetyltransferase (SoBAHD1) that attaches an acetyl group to TriF(Q)RXX to form saponin B, wherein the amino acid sequence of SoBAHD1 has at least 50% sequence identity to SEQ ID NO:

36.

19. An isolated polypeptide comprising: (i) a SobAS amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8; (ii) a SoC28 oxidase amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2; (iii) a SoC16C28 oxidase amino acid sequence having at least 50% sequence identity to SEQ ID NO: 4; (iv) a SoC23 oxidase amino acid sequence having at least 50% sequence identity to SEQ ID NO: 6; (v) a SoCSL amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10; (vi) a SoC3Gal amino acid sequence having at least 50% sequence identity to SEQ ID NO: 12; (vii) a SoQA-RXylT amino acid sequence having at least 50% sequence identity to SEQ ID NO: 14; (viii) a SoC28Fu amino acid sequence having at least 60% sequence identity to SEQ ID NO: 16; (ix) a SoC28Rha amino acid sequence having at least 50% sequence identity to SEQ ID NO: 18; (x) a SoC28Xyl1 amino acid sequence having at least 50% sequence identity to SEQ ID NO: 20; (xi) a SoC28Xyl2 amino acid sequence having at least 50% sequence identity to SEQ ID NO: 22; (xii) a SoGH1 amino acid sequence having at least 50% sequence identity to SEQ ID NO: 34; and / or (xiii) a SoBAHD1 amino acid sequence having at least 50% sequence identity to SEQ ID NO:

36.

20. An isolated nucleic acid encoding one or more polypeptides according to claim 19.

21. A vector comprising the nucleic acid according to claim 20.

22. A host cell comprising the nucleic acid according to claim 20 or the vector according to claim 21.

23. A method for producing a host cell, comprising: The host cell is transformed or transfected with a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a plurality of nucleotide sequences according to any one of claims 7 to 18 and 20.

24. The method according to claim 23, wherein: The host cell is a plant cell.

25. A method for producing a transgenic plant, the method comprising the steps of: (a) performing the method as claimed in claim 24; and (b) regenerating a plant from the transformed plant cell.

26. A transgenic plant, which can be obtained by the method according to claim 25, or is a clone of the transgenic plant, or a self-fertilized or hybrid progeny or other offspring, in, The expression of the heterologous nucleic acid enables the transgenic plant to have a higher ability to carry out the triterpene biosynthesis compared to the corresponding wild-type plant.

27. A method for producing triterpenes in a heterologous host, the method comprising: Cultivating the host cell according to any one of claims 13 to 18 and 22, and purifying the triterpene obtained therefrom.

28. A method for producing triterpenes in a heterologous host, the method comprising: The plant according to claim 26 is grown, then harvested, and the triterpenes obtained therefrom are purified.

29. The method according to claim 27 or 28, wherein: The triterpenoid is saponic acid or glycosylated saponic acid.

30. The method of claim 29, wherein: The glycosylated saponin is QA-Tri, QA-TriFRXX or QA-TriF(Q-Ac)RXX.

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