Methods and compositions related to the synthesis of QS-7 molecules

Abstract

The present invention relates to the biosynthetic pathway to the precursor of the QS-7 molecule, as well as the production pathway of the QS-7 molecule, the enzymes involved, the products produced, and the uses of the products.

Description

Technical Field

[0001] The present invention relates to the biosynthetic pathway to the precursor of the QS-7 molecule, as well as the manufacturing pathway of the QS-7 molecule, the enzymes involved, the products produced, and the uses of the products.

Background Art

[0002] QS-7 is a natural saponin extracted from the bark of Quillaja saponaria, a tree known as "soapbark" in Chile. The QS-7 extract was originally identified as a purified fraction of the crude bark extract of Quillaja Saponaria Molina obtained by RP-HPLC purification (peak 7) (Kensil et al. 1991). The QS-7 molecule incorporates a central triterpene core skeleton (quillaic acid), a branched trisaccharide is attached to the triterpene C-3 oxygen functional group, and a sugar chain is attached to the triterpene C-28 carboxyl group. The structures of the sugar chains at the C-28 position are different between QS-7 and QS-21 (see Figure 1). The QS-21 structure is represented by a linear tetrasaccharide consisting of fucose, rhamnose, xylose, and xylose (or apiose) as the terminal sugar. The QS-7 structure contains the same linear tetrasaccharide with apiose as the terminal sugar, and further incorporates two additional sugars (resulting in a branched hexasaccharide): (i) a rhamnose residue is incorporated at the C-3 position of the fucose residue of the linear tetrasaccharide, and (ii) a glucose residue is incorporated at the C-3 position of the rhamnose residue of the linear tetrasaccharide. A further difference between the two enzymes is that instead of incorporating an acyl chain into the fucose residue (QS-21), QS-7 incorporates an acetyl moiety at the C-4 position of this sugar residue (see Figure 1).

[0003] Saponins from Quillaja saponaria containing QS-7 have long been known to have a potent immunostimulatory effect that enhances antibody production and specific T cell responses. QS-7 exhibits similar potency to QS-21 and has reduced toxicity (Kensil et al. 1991). Due to these properties, Quillaja saponin-based adjuvants for vaccines have been developed. Of particular note, QS-7 is present in Novavax's "Matrix-M" (a part of the saponin fraction called "Fraction A" - see, for example, WO 2017 / 161151) and is utilized in the COVID-19 vaccine NVX-CoV2373.

[0004] The present invention describes methods for synthesizing precursors of the QS-7 molecule, the QS-7 molecule itself, and its variants, other than purification from wild Q. saponaria plants. The present invention also describes products useful as adjuvants in vaccine formulations. The present invention also relates to methods for producing such products, vectors, host cells, and enzymes involved in biological systems.

Summary of the Invention

[0005] The present invention relates to the formation of a branched-chain acetylated hexasaccharide of the QS-7 molecule. In particular, (i) a glucose (G) residue at the C-3 position of the rhamnose residue of the linear tetrasaccharide chain at the C-28 position of QA, (ii) a rhamnose (R) residue at the C-3 position of the D-fucose (F) of the linear tetrasaccharide chain at the C-28 position of QA, and (iii) the addition of an acetyl (Ac) moiety at the C-4 position of the D-fucose (F) of the linear tetrasaccharide chain at the C-28 position of QA (see Figure 1). The resulting QA derivatives are collectively designated as QA-tri(X / R)-F * -GR-Ac.

[0006] For the sake of simplicity, in the remainder of this specification, the linear tetrasaccharide chain at the C-28 position of QA and its precursors (i.e., sugar chains having only 2 or 3 sugars at the C-28 position) are represented as F * . Thus, in the present invention, "F *」 is understood as FR, FRX, FRXA, and / or FRXX (more simply, FRXA and FRXX may be denoted as FRX(X / A); see the list of abbreviations in this specification).

[0007] This invention includes biosynthetic preparations of QA-tri(X / R)-F * -GR-Ac and its precursors. The present invention also relates to the use of the QS-7 molecule (QA-triX-FRXA-GR-Ac) and its precursors and variants, such as QA-tri(X / R)-F * -GR-Ac, for example, its use as an adjuvant.

[0008] QA synthesis QA is a simple triterpene derived from β-amyrin, which is synthesized by cyclization of the universal linear precursor 2,3-oxidosqualene (OS) by oxidosqualene cyclase (OSC). This biosynthesis is known in the art and is described in WO2019 / 122259 etc., the content of which is incorporated by reference. This β-amyrin skeleton is further oxidized by a series of three cytochrome P450 monooxygenases with carboxylic acid, alcohol and aldehyde at positions C-28, C-16α and C-23 respectively to form kiralic acid (QA). OSC and C-28, C16α and C-23 oxidases are called QsbAS (β-amyrin synthase), QsCYP716-C-28, QsCYP716-C-16α and QsCYP714-C-2 oxidase respectively. Its biosynthetic pathway is shown in Figure 2.

[0009] C-3 branched-chain trisaccharide synthesis The C-3 branched trisaccharide chain is initiated by a D-glucopyranuronic acid (D-GlcpA) residue β-linked to the C-3 position of the QA backbone. Two sugars are attached to the D-GlcpA residue: a D-galactopyranose (D-Galp) residue linked by a β-1,2-bond and a D-xylopyranose (D-Xylp) moiety or an L-rhamnopyranose (L-Rhap) residue linked by a β-1,3-bond or an α-1,3-bond. The "scheme" of the glycosylation of QA to 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid}-cholic acid (QA-triR) or 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid}-cholic acid (QA-triX) is shown in Figure 3.

[0010] Seven enzymes with activities related to the production of QA 3-O trisaccharide (QA-triX or QA-triR) have been identified and are described in WO2020 / 260475 etc., the content of which is incorporated by reference. This includes two functionally redundant glucuronosyltransferases CSL1 and CslG2 that can add the first β-D-glucopyranuronic acid moiety to the C-3 position of cholic acid; a galactosyltransferase Qs-3-O-GalT that adds a β-D-galactopyranose residue to the C-2 position of β-D-glucopyranuronic acid; a xylosyltransferase Qs_0283870 that adds a β-D-xylopyranose residue to the C-3 position of β-D-glucopyranuronic acid; two rhamnosyltransferases DN20529_c0_g2_i8 and Qs_0283850 that add an α-L-rhamnopyranose residue to the C-3 position of β-D-glucopyranuronic acid; and a bifunctional enzyme Qs-3-O-RhaT / XylT that can add either a β-D-xylopyranose residue or an α-L-rhamnopyranose residue to the C-3 position of β-D-glucopyranuronic acid (see Figure 3).

[0011] For the sake of simplicity, in the present application, QA derivatives containing a branched trisaccharide at the C-3 position may be denoted as "QA-triX", "QA-triR", or "QA-tri(X / R)" (see the list of abbreviations).

[0012] C-28 linear tetrasaccharide synthesis F * is initiated by attaching a D-fucose residue to the C-28 position of the QA backbone via a β-linkage. In this step, an L-rhamnose residue is attached to the C-2 position of the fucose residue via an α-linkage, followed by attaching a D-xylose residue to the C-4 position of the rhamnose residue via a β-linkage. Finally, a D-xylose residue or a D-apiose residue is attached to the C-3 position of the xylose residue via a β-linkage.

[0013] As reported in PCT / EP2021 / 087323, ten enzymes with activities related to the production of F * have been identified. These include Qs-28-O-FucT (SEQ ID NO: 2) that transfers a D-fucose residue to the C-28 position of the QA backbone via a β-linkage; Qs-28-O-RhaT (SEQ ID NO: 4) that transfers an L-rhamnose residue to the D-fucose moiety; Qs-28-O-XylT3 (SEQ ID NO: 6) that transfers a D-xylose moiety to the L-rhamnose residue; Qs-28-O-XylT4 (SEQ ID NO: 8) that attaches a β-D-xylose residue to a β-D-xylose residue; Qs-28-O-ApiT4 (SEQ ID NO: 10) that attaches a β-D-apiose residue to a β-D-xylose residue (Figure 4). QsFucSyn (SEQ ID NO: 12) and QsFucSyn-like enzymes, which are oxidoreductases such as QsFSL-1 (SEQ ID NO: 48), QsFSL-2 (SEQ ID NO: 50), or SoFSL-1 (SEQ ID NO: 52) that can increase the production of UDP-D-fucose and / or reduce the 4-keto group of 4-keto-6-deoxy-glucose after addition to the QA backbone, have also been identified, and these have activities related to the production of F * . QsAXS1 (SEQ ID NO: 14), a UDP-α-D-apiose / UDP-xylose synthase that enhances the activity of the apiosyltransferase by increasing the availability of UDP-α-D-apiose, has also been previously identified.

[0014] Synthesis of C-28 acetylated branched hexasaccharide In the present invention, for the first time, the addition of a glucose residue at the C-3 position of the rhamnose residue of F for forming the QS-7 molecule and its precursors and variants, F * the addition of a rhamnose residue at the C-3 position of D-fucose of F, and F * the addition of a rhamnose residue at the C-3 position of D-fucose of F, and F * the biosynthetic pathway for the addition of an acetyl moiety at the C-4 position of D-fucose of F are described. As a result, the QS-7 molecule has an acetyl moiety at the C-4 position of the D-fucose residue of F and contains a hexasaccharide chain branched at the C-28 position (see Figure 1). * Therefore, the present invention provides a method for producing QS-7 and its precursors and variants. Also provided are the enzymes used in this method, polynucleotides encoding the enzymes, vectors containing the polynucleotides, host cells transformed with the vectors, and the use of the QS-7 molecule, its precursors and variants as adjuvants.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

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[0017] The first form of the present invention is a method for producing QA-tri(X / R)-F * -GR-Ac, wherein the acetyl (Ac) moiety is attached to the C-4 position of the D-fucose of F * and the rhamnose (R) residue is attached to the C-3 position of the D-fucose of F * and the glucose (G) residue is attached to the C-3 position of the rhamnose residue of F * This method involves QA-tri(X / R)-F * using i. The enzyme kiralic acid 28-O-fucosido[1,2]-rhamnosido[1,3]glucosyltransferase (QS-7-GlcT) having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. One or more enzymes selected from the enzyme kiralic acid 28-O-fucosido[1,4]acetyltransferase (QS-7-AcetylT) having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme (3S,5S,6S)-3,5-dihydroxy-6-methyloctanoyl-CoA transferase 9 (DMOT9) having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. The enzyme kiralic acid 28-O-fucosido[1,3]rhamnosyltransferase (QS-7-RhaT) having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58, in combination with QA-tri(X / R)-F* -GR-Ac formation. In this form of the present invention, QA is a chiral acid; Tri(X / R) is a branched trisaccharide at the C-3 position of the QA backbone terminated by either a xylose residue (X) or a rhamnose residue (R); F * is a disaccharide of a β-D-fucose residue (F) and R, also represented as FR; a trisaccharide of F, R, and X, also represented as FRX; a tetrasaccharide of F, R, X, and X, also represented as FRXX; or a tetrasaccharide of F, R, X, and a β-D-apiose residue (A), also represented as FRXX; G is a glucose residue; and Ac is an acetyl moiety.

[0018] A second form of the present invention is a method for producing biosynthetic QA-tri(X / R)-F * -GR-Ac in a host. This method includes the following steps: In the host a) Expressing the genes necessary for the biosynthesis of QA-triR-F * and / or QA-triX-F * and b) Introducing a polynucleotide encoding the following: i. The enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. One or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64, or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. Enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58. In this form of the present invention, QA is a kiralic acid; Tri(X / R) is a branched trisaccharide at the C-3 position of the QA backbone terminated by either a xylose residue (X) or a rhamnose residue (R); F * is a disaccharide of a β-D-fucose residue (F) and R, also represented as FR; a trisaccharide of F, R and X, also represented as FRX; a tetrasaccharide of F, R, X and X, also represented as FRXX; or a tetrasaccharide of F, R, X and a β-D-apiose residue (A), also represented as FRXA; G is a glucose residue; and Ac is an acetyl moiety.

[0019] The third form of the present invention is a method for producing biosynthetic QA-TriX-F * -GR-Ac. This method includes the following steps: In a host a) Expressing the genes necessary for the biosynthesis of QA-TriX-F * and b) Introducing a polynucleotide encoding the following: i. Enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. One or more enzymes selected from enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64, or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. Enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58. In this form of the present invention, QA-triX is 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-kijic acid; F * is a disaccharide of β-D-fucose residue (F) and rhamnose residue (R), also represented as FR; a trisaccharide of F, R and xylose residue (X), also represented as FRX; a tetrasaccharide of F, R, X and X, also represented as FRXX, or a tetrasaccharide of F, R, X and β-D-apiose residue (A), also represented as FRXA; G is a glucose residue; and Ac is an acetyl moiety.

[0020] In the third form of the present invention, F * can be FRXA. Accordingly, the present invention includes a method for producing biosynthetic QA-triX-FRXA-GR-Ac in a host. The method includes the following steps: in the host a) expressing the genes necessary for the biosynthesis of QA-triX-FRXA, and b) introducing a polynucleotide encoding: i. Enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. One or more enzymes selected from enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64, or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. Enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58.

[0021] The fourth aspect of the present invention is a method for producing biosynthetic QA-triR-F * -GR-Ac in a host. This method includes the following steps: a) Expressing the genes necessary for the biosynthesis of QA-triR-F * and b) A polynucleotide encoding the following: i. Enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. Enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, one or more enzymes selected from, and iii. Enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58 introducing into the host. In this aspect of the present invention, QA-triR is 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronosyl}-kijic acid; F * is a disaccharide of β-D-fucose residue (F) and rhamnose residue (R), also represented as FR; a trisaccharide of F, R and xylose residue (X), also represented as FRX; a tetrasaccharide of F, R, X and X, also represented as FRXX; a tetrasaccharide of F, R, X and β-D-apiose residue (A), also represented as FRXA; G is a glucose residue; and Ac is an acetyl moiety.

[0022] In a first form of the present invention, steps (i), (ii) and (iii) may occur in that order. QA-Tri(X / R)-F * is first combined with an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56, to form QA-Tri(X / R)-F * -G. In particular, F * can be FRX. F * may be FRX(X / A). Next, QA-Tri(X / R)-F * -G is combined with one or more enzymes selected from an enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, an enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or an enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64, or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, to form QA-Tri(X / R)-F * -G-Ac. In particular, F * can be FRX. F * may be FRX(X / A). Finally, QA-Tri(X / R)-F * -G-Ac is combined with an enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58, to form QA-Tri(X / R)-F * -GR-Ac. In particular, F * can be FRX. F * may be FRX(X / A). In steps (i), (ii) and (iii), F * can be FRX.

[0023] The steps of this form of the present invention may occur in the order of (ii), (iii), and then (i). QA-Tri(X / R)-F * is combined with one or more enzymes selected from the group consisting of an enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, an enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62, an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or an enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64, or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64 to form QA-Tri(X / R)-F * -Ac. In particular, F * can be FR. F * may be FRX. F * may be FRX(X / A). Next, QA-Tri(X / R)-F * -Ac is combined with an enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58 to form QA-Tri(X / R)-F * -R-Ac. In particular, F * can be FR. F * may be FRX. F * may be FRX(X / A). Finally, QA-Tri(X / R)-F * -R-Ac is combined with an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56 to form QA-Tri(X / R)-F * -GR-Ac. F * can be FRX. F * may be FRX(X / A). In steps (i) and (ii), F * can be FR, and in step (iii), F * may be FRX.

[0024] The steps of this embodiment of the present invention may occur in the order of (ii), (i), and then (iii). QA-Tri(X / R)-F * is combined with one or more enzymes selected from an enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, an enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or an enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64 to form QA-Tri(X / R)-F * -Ac. F * may be FR. F * may be FRX. F * may be FRX(X / A). Next, QA-Tri(X / R)-F * -Ac is combined with an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56 to form QA-Tri(X / R)-F * -G-Ac. F * may be FRX. F * may be FRX(X / A). Finally, QA-Tri(X / R)-F * -G-Ac is combined with an enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58 to form QA-Tri(X / R)-F * -GR-Ac. F * may be FRX. F * may be FRX(X / A). In step (i), F * may be FR, and in steps (ii) and (iii), F * may be FRX.

[0025] In the first embodiment of the present invention, Tri(X / R) may be TriX, and F *It may be FRXA. Accordingly, the present invention includes a method for producing QA-triX-FRXA-GR-Ac, wherein the acetyl (Ac) moiety is attached to the C-4 position of the D-fucose of FRXA, the rhamnose (R) residue is attached to the C-3 position of the D-fucose of FRXA, and the glucose (G) residue is attached to the C-3 position of the rhamnose residue of FRXA. The method comprises i. an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. an enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60; an enzyme having the amino acid sequence of SEQ ID NO: 62, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or an enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64, or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. an enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58, in combination to form QA-triX-FRXA-GR-Ac.

[0026] When tri(X / R) is triX and F * is FRXA, the steps of the first form of the present invention may occur in the order of (i), (ii), and then (iii). These steps may also occur in the order of (ii), (iii), and then (i). These steps may also occur in the order of (ii), (i), and then (iii).

[0027] As defined herein, F * may be FR, FRX, FRXA, and / or FRXX. F * The sugar of the F chain is added to the C-28 position of QA-tri(X / R). F *When it is a mixture containing FRXX and FRXA, the ratio of FRXX to FRXA can vary. The ratio of FRXX to FRXA in the mixture can vary in percentage. Preferably, the mixture contains, for example, 10 - 90% of FRXX such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and, for example, 90% - 10% of FRXA such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%. Preferably, the mixture contains 60% of FRXX and 40% of FRXA, or 50% each.

[0028] In an embodiment where the acetyl (Ac) moiety is attached to the C-4 position of D-fucose of F * F is FR, FRX, FRXA and / or FRXX. In particular, F * can be FRXA. Similarly, in an embodiment where QA-tri(X / R)-F * is combined with one or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64 to form QA-tri(X / R)-F * -Ac, F * is FR, FRX, FRXA and / or FRXX. In particular, F * can be FRXA. QA-tri(X / R)-F * can be FRXA. QA-tri(X / R)-F *-G is combined with one or more enzymes selected from an enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, an enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or an enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, QA-tri(X / R)-F * -In an embodiment where -G-Ac is formed, F * is FRX, FRXA and / or FRXX, particularly F * can be FRXA.

[0029] F * -In an embodiment where a rhamnose (R) residue is bonded to the C-3 position of the D-fucose of F * is FR, FRX, FRXA and / or FRXX, and the acetyl moiety must be bonded to the C-4 position of the D-fucose of F * . QA-tri(X / R)-F * -Ac is combined with an enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58, QA-tri(X / R)-F * -In an embodiment where -R-Ac is formed, F * is FR, FRX, FRXA and / or FRXX. Particularly F * can be FRXA. QA-tri(X / R)-F * -G-Ac is combined with an enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58, QA-tri(X / R)-F * -In an embodiment where -GR-Ac is formed, F * is FRX, FRXA and / or FRXX. Particularly F * can be FRXA.

[0030] In an embodiment where a glucose (G) residue is attached to the C-3 position of the rhamnose moiety of F * , F * is FRX, FRXA and / or FRXX. In particular, F * can be FRXA. QA-tri(X / R)-F * is combined with an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56, and in an embodiment where QA-tri(X / R)-F * -G is formed, F * is FRX, FRXA and / or FRXX. In particular, F * can be FRXA. QA-tri(X / R)-F * -Ac is combined with an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56, and when QA-tri(X / R)-F*-G-Ac is formed, F * is FRX, FRXA and / or FRXX. In particular, F * can be FRXA. QA-tri(X / R)-F * -R-Ac is combined with an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56, and in an embodiment where QA-tri(X / R)-F * -GR-Ac is formed, F * is FRX, FRXA and / or FRXX. In particular, F * can be FRXA.

[0031] As defined herein, the QA-tri(X / R)-F * derivative can be QA-tri(X / R)-FR, QA-tri(X / R)-FRX, QA-tri(X / R)-FRXX, QA-tri(X / R)-FRXA, QA-triR-FR, QA-triR-FRX, QA-triR-FRXX, QA-triR-FRXA, QA-triX-FR, QA-triX-FRX, QA-triX-FRXX or QA-triX-FRXA.

[0032] When the QA-trisaccharide (X / R) is a mixture containing QA-trisaccharide X and QA-trisaccharide R, the ratio of QA-trisaccharide X to QA-trisaccharide R can vary. The ratio of QA-trisaccharide X to QA-trisaccharide R in the mixture can vary in percentage. Preferably, the mixture contains, for example, 10% to 90% of QA-trisaccharide X such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and, for example, 90% to 10% of QA-trisaccharide R such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%.

[0033] Synthesis of C-28 acetylated branched-chain hexasaccharide The QS-7 molecule attaches a glucose residue to the C-3 position of the rhamnose residue of F * , attaches a rhamnose residue to the C-3 position of the D-fucose of F * , and incorporates an acetyl moiety at the C-4 position of the D-fucose of F * The inventors have identified in vitro and in vivo the enzymes that add the glucose residue, rhamnose residue, and acetyl moiety to the required positions of the core molecule. "Core molecule" means one or more of the following QA derivatives: QA-trisaccharide X-FR, QA-trisaccharide X-FRX, QA-trisaccharide X-FRXX, QA-trisaccharide X-FRXA, QA-trisaccharide R-FR, QA-trisaccharide R-FRX, QA-trisaccharide R-FRXA, QA-trisaccharide R-FRXX, QA-trisaccharide (X / R)-FR, QA-trisaccharide (X / R)-FRX, QA-trisaccharide (X / R)-FRXA, QA-trisaccharide (X / R)-FRXX.

[0034] In the method of the present invention, the steps of adding the glucose and rhamnose residues and the acetyl moiety can be performed in a specific order, or in any order, or simultaneously.

[0035] In the method of the present invention, the transfer of the acyl moiety to the C-4 position of the D-fucose of F * (for example, to form QA-trisaccharide (X / R)-F * -Ac from QA-trisaccharide (X / R)-F *The transfer of the acyl moiety to ( ) can be carried out by the enzyme QS-7-AcetylT (SEQ ID NO: 60), or an enzyme having at least 70% sequence identity to the sequence of QS-7-AcetylT, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64. These enzymes can transfer an acyl group to the C-4 position of the D-fucose of the F * chain. The function of the enzyme can be determined as described in Example 2, for example.

[0036] The function of QS-7-AcetylT, SOAP10 or DMOT9 can be determined by expressing, in a heterologous host such as N. benthamiana or yeast, the enzyme necessary to generate QA-tri(X / R)-F * and the candidate QS-7-AcetylT, SOAP10 or DMOT9. The presence of the expected product can be evaluated by LC-MS analysis and ultimately complemented by NMR analysis. Alternatively, QA-tri(X / R)-F * is purified from a plant extract, or an in vitro test is preferably performed that generates in vitro, either containing the glycosyltransferase necessary for the formation of kiralic acid and QA-tri(X / R)-F * or containing β-amylase and the enzyme necessary for the formation of QA-tri(X / R)-F * . The activity of the candidate QS-7-AcetylT, SOAP10 or DMOT9 is then tested against the QA-tri(X / R)-F * substrate, and the formation of the product is determined by LC-MS analysis.

[0037] Throughout this specification, when referring to an enzyme (SEQ ID NO:), this refers to the enzyme according to that SEQ ID NO:. For example, "QS-7-AcetylT (SEQ ID NO: 60)" means the enzyme QS-7-AcetylT according to SEQ ID NO: 60.

[0038] The enzyme used in the present invention may contain one or more conservative amino acid substitutions, and the resulting enzyme has a similar amino acid sequence and / or retains the same function. Those skilled in the art will recognize that various amino acids have similar biochemical properties and are thus "conservative". One or more such amino acids of a protein (such as an enzyme), polypeptide or peptide can often be substituted with one or more other such amino acids without eliminating the desired activity of the protein, polypeptide or peptide.

[0039] Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted with one another (amino acids with aliphatic side chains). Among these possible substitutions, it is preferred to use glycine and alanine substituted with each other (since both have relatively short side chains), and to use valine, leucine and isoleucine substituted with each other (since both have large hydrophobic aliphatic side chains). Other amino acids often substituted with one another include the following: phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); lysine, arginine and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). It should be understood that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, the methyl group of alanine can be substituted with an ethyl group and / or minor modifications can be made to the peptide backbone. It is preferred that only L-amino acids are present, whether natural or synthetic amino acids are used. This type of substitution is often referred to as a "conservative" amino acid substitution.

[0040] In the art, "identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, and is determined by comparing the sequences. In the art, identity refers to the degree of sequence similarity between polypeptide or polynucleotide sequences, as the case may be, and is determined by the match of the strings of the said sequences. There are numerous methods for measuring the identity between two polypeptides or two polynucleotide sequences, but the methods generally used for determining identity are incorporated into computer programs. Preferred computer programs for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0041] For the comparison of amino acid sequences, programs such as the CLUSTAL program can be used. This program compares amino acid sequences and inserts spaces into either sequence as appropriate to find the optimal alignment. It is possible to calculate the amino acid identity or similarity (identity + conservation of amino acid type) for the optimal alignment. Programs such as BLASTx align the longest parts of similar sequences and assign values to the matches. As a result, multiple similar regions can be found, and comparison results with different scores can be obtained.

[0042] The percentage of identity between two amino acid sequences or two polynucleotide sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for the best alignment with the second sequence), and then comparing the corresponding amino acid residues or nucleotides at the corresponding positions. "Best alignment" refers to an alignment of two sequences that results in the highest percentage of identity. The percentage of identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions × 100).

[0043] The percent identity between two arrays can be determined using mathematical algorithms well known to those skilled in the art. Examples of mathematical algorithms for comparing two arrays that can be used include the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. In a BLAST nucleotide search, when the NBLAST program is run with a score = 100 and word length = 12, a nucleotide sequence homologous to the nucleic acid molecule can be obtained. In a BLAST protein search, when the XBLAST program is run with a score = 50 and word length = 3, an amino acid sequence homologous to the protein molecule used in the present invention can be obtained. To obtain a gap alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches to detect remote relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST or NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, incorporates such an algorithm.Other algorithms for sequence analysis known to those skilled in the art include ADVANCE and ADAM described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10: 3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0044] Mutations such as conservative substitutions, insertions, deletions, etc. may be introduced into the sequence using any suitable method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme cloning, or ligation-independent cloning (LIC) procedures. These methods are described in detail in many standard molecular biology texts. For details of polymerase chain reaction (PCR) and restriction enzyme cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3 rd Ed.) CSHL Press. Further details of ligation-independent cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6.

[0045] In the method of the present invention, F *The transfer of the acyl moiety to the C-4 position of D-fucose may be carried out by the enzyme QS-7-AcetylT (SEQ ID NO: 60), or an enzyme having at least 70% sequence identity to the sequence of QS-7-AcetylT (SEQ ID NO: 60). The amino acid sequence of the QS-7-AcetylT enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 56. Thus, in some embodiments, QS-7-AcetylT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 60. For the enzymes defined herein with respect to sequence identity, typically, F * retains the function of transferring an acyl unit to the C-4 position of D-fucose.

[0046] F * The transfer of the acyl moiety to the C-4 position of D-fucose may be carried out by the enzyme SOAP10 (SEQ ID NO: 62), or an enzyme having at least 70% sequence identity to the sequence of SOAP10 (SEQ ID NO: 62). The amino acid sequence of the SOAP10 enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 62. Thus, in some embodiments, SOAP10 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 62. For the enzymes defined herein with respect to sequence identity, typically, D-fucose F * retains the function of transferring an acyl unit to the C-4 position.

[0047] F *The transfer of an acyl moiety to the C-4 position of D-fucose may be carried out by the enzyme DMOT9 (SEQ ID NO: 64), or an enzyme having at least 25% sequence identity to the sequence of DMOT9 (SEQ ID NO: 64). The amino acid sequence of the DMOT9 enzyme may have at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 64. Thus, in some embodiments, the DMOT9 enzyme has at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 64, preferably at least 90%, more preferably at least 95% identity. For enzymes as defined herein with respect to sequence identity, typically, F * retains the function of transferring an acyl unit to the C-4 position of D-fucose.

[0048] The percentage sequence identity discussed in this application is the percentage sequence identity over the full length of the sequence defined by the SEQ ID NO. This may include truncated sequences having the same sequence identity measured over the length of the truncated sequence. The truncated sequence may have the same homology as the percentage sequence identity of the SEQ ID NO, regardless of the length of the truncated sequence. The truncated sequence may be at least half the length of the full-length sequence, preferably at least three-quarters the length of the full-length sequence.

[0049] In the method of the present invention, F * the transfer of a rhamnose residue to the C-3 position of D-fucose may be carried out by the enzyme QS-7-RhaT (SEQ ID NO: 58), or an enzyme having at least 70% sequence identity to the sequence of QS-7-RhaT. This enzyme can transfer a rhamnose moiety to the C-3 position of D-fucose in the F * chain. The function of this enzyme can be determined as described, for example, in Example 3.

[0050] The function of QS-7-RhaT can be determined by expressing QS-7-RhaT, which is a candidate for the enzyme necessary to generate QA-tri(X / R)-F * -Ac in heterologous hosts such as N. benthamiana and yeast. The presence of the expected product can be evaluated by LC-MS analysis and ultimately complemented by NMR analysis. Alternatively, QA-tri(X / R)-F * can be purified from plant extracts or an in vitro test may be preferred where it is generated in vitro in an assay containing the sugar glycosyltransferase necessary for the production of QA-tri(X / R)-F * or containing β-amylase and the enzyme necessary for the production of QA-tri(X / R)-F * . The activity of the candidate QS-7-AcetylT is then tested against the QA-tri(X / R)-F * substrate and the formation of the product is determined by LC-MS analysis.

[0051] In the method of the present invention, the transfer of a rhamnose residue to the C-3 position of D-fucose of F * may be carried out by the enzyme QS-7-RhaT (SEQ ID NO: 58) or an enzyme having at least 70% sequence identity to the sequence of QS-7-RhaT (SEQ ID NO: 58). The amino acid sequence of the QS-7-RhaT enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 58. Thus, in some embodiments, QS-7-RhaT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 58. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of transferring a rhamnose moiety to the C-3 position of D-fucose of F * .

[0052] In the method of the present invention, QA-tri(X / R)-F *QA-tri(X / R)-F for forming -GR-Ac * The transfer of a glucose residue to a molecule containing -R-Ac may be carried out by the enzyme QS-7-GlcT (SEQ ID NO: 56), or an enzyme having at least 70% sequence identity to SEQ ID NO: 5. These enzymes can transfer a glucose residue to the C-3 position of the rhamnose residue of F * The function of the enzyme can be determined as described, for example, in Example 1.

[0053] The function of QS-7-GlcT can be determined by expressing QA-tri(X / R)-F * and the enzyme necessary to produce -R-Ac and the candidate QS-7-GlcT in a heterologous host such as N. benthamiana or yeast. The presence of the expected product can be evaluated by LC-MS analysis and ultimately complemented by NMR analysis. Alternatively, QA-tri(X / R)-F * -R-Ac can be purified from plant extracts, or contain the glycosyltransferase necessary for the production of kiralic acid and QA-tri(X / R)-F * -R-Ac, or an in vitro test that produces in vitro in an assay containing β-amylase and the enzyme necessary for the production of QA-tri(X / R)-F * -R-Ac may also be preferred in some cases. The activity of the candidate QS-7-GlcT is tested against the QA-tri(X / R)-F * -R-Ac substrate, and the formation of the product is determined by LC-MS analysis.

[0054] In the method of the present invention, QA-tri(X / R)-F * QA-tri(X / R)-F for forming -GR-Ac *The transfer of a glucose residue to a molecule containing -R-Ac may be performed by the enzyme QS-7-GlcT (SEQ ID NO: 56), or an enzyme having at least 70% sequence identity to the sequence of QS-7-GlcT (SEQ ID NO: 56). The amino acid sequence of the QS-7-GlcT enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 56. Thus, in some embodiments, QS-7-GlcT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 56. For the enzymes defined herein with respect to sequence identity, typically, QA-tri(X / R)-F * transfer a glucose residue to a molecule containing -R-Ac and retain the function of forming QA-tri(X / R)-F * -GR-Ac.

[0055] The percentage sequence identity of the sequences to QS-7-RhaT, QS-7-GlcT, QS-7-AcetylT, DMOT9 and SOAP10 may all be the same or different.

[0056] As described above, the method of the present invention involves adding an acyl moiety, a glucose moiety and a rhamnose moiety to QA-tri(X / R)-F * as described above. QA-tri(X / R)-F * is described above. A further feature of the method of the present invention is the step for producing a branched-chain trisaccharide (QA-tri(X / R)) at the C-3 position of the QA backbone, a molecule containing the QA backbone, and a linear sugar chain (F * ) at the C-28 position of the molecule containing the QA backbone (QA-tri(X / R)-F * ).

[0057] QA synthesis QA-tri(X / R)-F *One step in the method of forming the QA backbone of a molecule comprising is the cyclization of 2,3-oxidosqualene to form a molecule comprising the triterpene β-amyrin. This step is carried out by an oxidosqualene cyclase. In particular, the oxidosqualene cyclase can be an enzyme that follows a sequence having at least 50% sequence identity to QsbAS (SEQ ID NO: 18). The oxidosqualene cyclase can be encoded by the polynucleotide sequence of SEQ ID NO: 17.

[0058] This step involves an oxidosqualene cyclase enzyme having at least 50% sequence identity to the sequence of QsbAS (SEQ ID NO: 18). The amino acid sequence of the QsbAS enzyme can have at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18. Thus, in some embodiments, QsbAS has at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 18. For enzymes as defined herein with respect to sequence identity, typically, they retain the function of forming a molecule comprising the triterpene β-amyrin by the cyclization reaction of 2,3-oxidosqualene.

[0059] Molecules containing a β - amyrin skeleton are further oxidized at positions C - 28, C - 16α, and C - 23 to carboxylic acid, alcohol, and aldehyde, respectively. Another step of this feature of the present invention is to form a carboxylic acid at the C - 28 position by oxidizing a molecule containing a β - amyrin skeleton. This step is carried out by cytochrome P450 monooxygenase. The cytochrome P450 monooxygenase is C - 28 oxygenase QsCYP716 - C - 28. For example, the C - 28 oxygenase QsCYP716 - C - 28 can be one that follows a sequence having at least 50% sequence identity to SEQ ID NO: 20. QsCYP716 - C - 28 can be encoded by the polynucleotide sequence of SEQ ID NO: 19 or a sequence having at least 50% sequence identity to SEQ ID NO: 19.

[0060] This step includes a cytochrome P450 monooxygenase having at least 50% sequence identity to the sequence of QsCYP716 - C - 28 (SEQ ID NO: 20). The amino acid sequence of the QsCYP716 - C - 28 enzyme can have at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20. Thus, in some embodiments, QsCYP716 - C - 28 has at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 20. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of forming a carboxylic acid at the C - 28 position by oxidizing a molecule containing a β - amyrin skeleton.

[0061] Another step of this feature of the present invention is to form an alcohol at the C-16 position by the oxidation of a molecule containing the β-amyrin skeleton. This step is carried out by cytochrome P450 monooxygenase. The cytochrome P450 monooxygenase is C-16α oxygenase QsCYP716-C-16α. For example, the C-16α oxygenase QsCYP716-C-16α can be one that follows a sequence having at least 50% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 22. QsCYP716-C-16α can be encoded by a polynucleotide sequence of SEQ ID NO: 21 or a sequence having at least 50% sequence identity to SEQ ID NO: 21.

[0062] This step involves a cytochrome P450 monooxygenase having at least 50% sequence identity to the sequence of QsCYP716-C-16α (SEQ ID NO: 22). The amino acid sequence of the QsCYP716-C-16α enzyme can have at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 22. Thus, in some embodiments, QsCYP716-C-16α has at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 22. For the enzymes defined herein with respect to sequence identity, typically, the function of forming an alcohol at the C-16 position by the oxidation of a molecule containing the β-amyrin skeleton is retained.

[0063] A further step of this feature of the present invention is to form an aldehyde at the C-23 position by oxidation of a molecule containing a β-amyrin skeleton. This step is carried out by cytochrome P450 monooxygenase. The cytochrome P450 monooxygenase is C-23 oxidase QsCYP714-C-23. For example, C-23 oxidase QsCYP714-C-23 can be one that follows a sequence having at least 50% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 24. QsCYP714-C-23 can be encoded by the polynucleotide sequence of SEQ ID NO: 23 or a sequence having at least 50% sequence identity to SEQ ID NO: 23.

[0064] This step involves a cytochrome P450 monooxygenase having at least 50% sequence identity to the sequence of QsCYP714-C-23 (SEQ ID NO: 24). The amino acid sequence of the QsCYP714-C-23 enzyme can have at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24. Thus, in some embodiments, QsCYP714-C-23 has at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 24. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of forming an aldehyde at the C-23 position by oxidation of a molecule containing a β-amyrin skeleton.

[0065] These steps form a QA skeleton.

[0066] This feature of the invention relates to a method for producing a molecule comprising a QA backbone, which comprises a number of steps. These steps can be carried out in a specific order, or in any order, or simultaneously. Preferably, this molecule is formed by sequentially oxidizing at the C-28, C-16α and C-23 positions following the generation of the β-amyrin backbone. The steps of this feature in this aspect of the invention are described for the above-described preferred situation. However, the steps may occur in any order.

[0067] C-3 branched-chain trisaccharide and F * The sugar units forming are then added. Preferably, a molecule comprising a QA backbone is produced, then the step of adding a C-3 chain is carried out, and then the step of adding F * is carried out. However, these steps can be carried out in a specific order, or in any order, or simultaneously.

[0068] C-3 branched-chain trisaccharide synthesis QA-tri(X / R)-F * The step of forming the tri(X / R) of the molecule comprising QA-tri(X / R)-F is described for the case where the C-3 branched-chain trisaccharide at the C-3 position of the molecule comprising a QA backbone is initiated by attaching a β-D-glucopyranuronic acid moiety to the molecule comprising QA, to form a molecule comprising QA-mono. However, these steps may occur in any order.

[0069] The first step in forming the C-3 chain is to attach a β-D-glucopyranuronic acid moiety to a molecule containing QA to form a molecule containing QA-mono. This step can be performed by the enzyme QsCSL1 according to SEQ ID NO: 26 or the enzyme QsCslG2 according to SEQ ID NO: 28, or by a sequence having at least 70% sequence identity to SEQ ID NO: 26 or 28. QsCSL1 can be encoded by the polynucleotide sequence of SEQ ID NO: 25 or a sequence having at least 70% sequence identity to SEQ ID NO: 25. QsCslG2 can be encoded by the polynucleotide sequence of SEQ ID NO: 27, or a sequence having at least 70% sequence identity to SEQ ID NO: 27.

[0070] This step includes enzymes having at least 70% sequence identity to the sequences of QsCSL1 and QsCslG2 (SEQ ID NO: 26 or 28, respectively). The amino acid sequence of the QsCSL1 enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26. The amino acid sequence of the QsCslG2 enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 28. Thus, in some embodiments, QsCSL1 and / or QsCslG2 have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 26 or 28. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of adding a β-D-glucopyranuronic acid moiety to a molecule containing QA to form a molecule containing QA-mono.

[0071] Another step in the method of forming the C-3 linkage is to add a D-galactopyranose moiety to the β-D-glucopyranuronic acid moiety on the molecule containing the QA-mono to form a molecule containing the QA-di. This step can be carried out by the enzyme Qs-3-O-GalT according to SEQ ID NO: 30 or a sequence having at least 70% sequence identity to SEQ ID NO: 30. Qs-3-O-GalT can be encoded by the polynucleotide sequence of SEQ ID NO: 29 or a sequence having at least 70% sequence identity to SEQ ID NO: 29.

[0072] This step encompasses enzymes having at least 70% sequence identity to the sequence of Qs-3-O-GalT (SEQ ID NO: 30). The amino acid sequence of the Qs-3-O-GalT enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 30. Thus, in some embodiments, Qs-3-O-GalT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 30. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of attaching a D-galactopyranose moiety to the β-D-glucopyranuronic acid moiety on the molecule containing the QA-mono to form a molecule containing the QA-di.

[0073] A further step in the method of forming the C-3 linkage is to add an L-rhamnopyranose moiety to the β-D-glucopyranuronic acid moiety on the molecule containing the QA-j to form a molecule containing the QA-tri R. This step can be performed by the enzyme DN20529_c0_g2_i8 of SEQ ID NO: 36, the enzyme Qs_0283850 of SEQ ID NO: 34, or the enzyme Qs-3-O-RhaT / XylT of SEQ ID NO: 3, or a sequence having at least 70% sequence identity to SEQ ID NO: 36, 34 or 32. DN20529_c0_g2_i8 can be encoded by the polynucleotide sequence of SEQ ID NO: 35 or a sequence having at least 70% sequence identity to SEQ ID NO: 35. Qs_0283850 can be encoded by the polynucleotide sequence of SEQ ID NO: 33 or a sequence having at least 70% sequence identity to SEQ ID NO: 33. Qs-3-O-RhaT / XylT can be encoded by the polynucleotide sequence of SEQ ID NO: 31 or a sequence having at least 70% sequence identity to SEQ ID NO: 31.

[0074] This step includes an enzyme having at least 70% sequence identity to the sequence of DN20529_c0_g2_i8, Qs_0283850, or Qs-3-O-RhaT / XylT (SEQ ID NO: 36, 34, or 32, respectively). The amino acid sequence of the DN20529_c0_g2_i8 enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36. The amino acid sequence of the Qs_0283850 enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 34. The amino acid sequence of the Qs-3-O-RhaT / XylT enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. Thus, in some embodiments, DN20529_c0_g2_i8, Qs_0283850, and / or Qs-3-O-RhaT / XylT have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 36, 34, or 32. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of adding an L-rhamnopyranose moiety to the β-D-glucopyranuronic acid moiety on a molecule containing QA-di to form a molecule containing QA-triR.

[0075] A further step in the method of forming the C-3 chain is to add a β-D-xylopyranose moiety to the β-D-glucopyranose moiety on the molecule containing QA-di to form a molecule containing QA-tri R. This step can be carried out by the enzyme Qs_0283870 of SEQ ID NO: 38, or the enzyme Qs-3-O-RhaT / XylT of SEQ ID NO: 32, or a sequence having at least 70% sequence identity to SEQ ID NO: 38 or 32. Qs_0283870 can be encoded by the polynucleotide sequence of SEQ ID NO: 37 or a sequence having at least 70% sequence identity to SEQ ID NO: 37. Qs-3-O-RhaT / XylT can be encoded by the polynucleotide sequence of SEQ ID NO: 31 or a sequence having at least 70% sequence identity to SEQ ID NO: 31.

[0076] This step encompasses enzymes having at least 70% sequence identity to the sequences of Qs_0283870 or Qs-3-O-RhaT / XylT (SEQ ID NOs: 38 or 32 respectively). The amino acid sequence of the Qs_0283870 enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 38. The amino acid sequence of the Qs-3-O-RhaT / XylT enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32. Thus, in some embodiments, Qs_0283870 and / or Qs-3-O-RhaT / XylT have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 38 or 32. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of binding a β-D-xylopyranose moiety to the β-D-glucopyranuronic acid moiety on the molecule containing QA-di to form a molecule containing QA-tri X.

[0077] These steps form the C-3 chain of the QA backbone.

[0078] Synthesis of C-28 linear tetrasaccharide QA-tri(X / R)-F * F of a molecule containing * The step of forming is the F of a molecule containing the QA backbone * is initiated by adding a UDP-α-D-fucose moiety to a molecule containing QA-tri(X / R) to form a molecule containing QA-tri(X / R)-F. However, these steps may occur in any order. For example, F * is generated and then may be attached to the QA-tri(X / R) backbone.

[0079] F * The first step of forming may be to add a UDP-α-D-fucose moiety to the C-28 position of a molecule containing QA-tri(R / X) to form a molecule containing QA-tri(R / X)-F. This step can be carried out by the enzyme Qs-28-O-FucT of a sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 2. Qs-28-O-FucT can be encoded by the polynucleotide sequence of SEQ ID NO: 1 or a sequence having at least 70% sequence identity to SEQ ID NO: 1. F * The first step of forming may be to add UDP-4-keto,6-deoxy-D-glucose to a molecule containing QA-tri(R / X) to form a molecule containing QA-tri(R / X)-F. This step can be carried out by the enzyme Qs-28-O-FucT of SEQ ID NO: 2 or a sequence having at least 70% sequence identity to SEQ ID NO: 2, and QsFucSyn of SEQ ID NO: 12 or a sequence having at least 45% sequence identity to SEQ ID NO: 12. Qs-28-O-FucT can be encoded by the polynucleotide sequence of SEQ ID NO: 1 or a sequence having at least 70% sequence identity to SEQ ID NO: 1. QsFucSyn can be encoded by the polynucleotide sequence of SEQ ID NO: 11 or a sequence having at least 45% sequence identity to SEQ ID NO: 11.

[0080] This step includes an enzyme having at least 70% sequence identity to the sequence of Qs-28-O-FucT (SEQ ID NO: 2). The amino acid sequence of the Qs-28-O-FucT enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2. Thus, in some embodiments, Qs-28-O-FucT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 2. For the enzymes defined herein with respect to sequence identity, typically, it has the function of adding a UDP-α-D-fucose moiety to the C-28 position of a molecule containing QA-tri(R / X) to form a molecule containing QA-tri(R / X)-F; or the function of adding UDP-4-keto,6-deoxy-D-glucose to a molecule containing QA-tri(R / X) to form a molecule containing QA-tri(R / X)-F.

[0081] This step also includes an enzyme having at least 45% sequence identity to the sequence of QsFucSyn (SEQ ID NO: 12). The amino acid sequence of the QsFucSyn enzyme can have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12. Thus, in some embodiments, QsFucSyn has at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 12. For the enzymes defined herein with respect to sequence identity, typically, it has the function of adding a UDP-α-D-fucose moiety to the C-28 position of a molecule containing QA-tri(R / X) to form a molecule containing QA-tri(R / X)-F; or the function of adding UDP-4-keto,6-deoxy-D-glucose to a molecule containing QA-tri(R / X) to form a molecule containing QA-tri(R / X)-F.

[0082] F* Another step for forming * is to add a UDP-β-L-rhamnose moiety to the UDP-α-D-fucose moiety on the molecule containing QA-tri(R / X)-F to form a molecule containing QA-tri(R / X)-FR. This step can be carried out by the enzyme Qs-28-O-RhaT of SEQ ID NO: 4 or a sequence having at least 70% sequence identity to SEQ ID NO: 4. Qs-28-O-RhaT can be encoded by the polynucleotide sequence of SEQ ID NO: 3 or a sequence having at least 70% sequence identity to SEQ ID NO: 3.

[0083] This step also includes enzymes having at least 70% sequence identity to the sequence of Qs-28-O-RhaT (SEQ ID NO: 4). The amino acid sequence of the Qs-28-O-RhaT enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4. Thus, in some embodiments, Qs-28-O-RhaT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 4. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of adding a UDP-β-D-rhamnose moiety to the UDP-α-D-fucose moiety on the molecule containing QA-tri(R / X) to form a molecule containing QA-tri(R / X)-FR.

[0084] F * A further step for forming * is to add a UDP-α-D-xylose moiety to the UDP-β-L-rhamnose moiety on the molecule containing QA-tri(R / X)-FR to form a molecule containing QA-tri(R / X)-FRX. This step can be carried out by the enzyme Qs-28-O-XylT3 according to SEQ ID NO: 6 or a sequence having at least 70% sequence identity to SEQ ID NO: 6. Qs-28-O-XylT3 can be encoded by the polynucleotide sequence of SEQ ID NO: 5 or a sequence having at least 70% sequence identity to SEQ ID NO: 5.

[0085] This step also includes an enzyme having at least 70% sequence identity to the sequence of Qs-28-O-XylT3 (SEQ ID NO: 6). The amino acid sequence of the Qs-28-O-XylT3 enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6. Thus, in some embodiments, Qs-28-O-XylT3 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 6. For the enzymes defined herein with respect to sequence identity, typically, the function of adding a UDP-α-D-xylose moiety to the UDP-β-L-rhamnose moiety on a molecule containing QA-tri(R / X)-FR to form a molecule containing QA-tri(R / X)-FRX is retained.

[0086] F * Any step for forming F is to add a UDP-α-D-xylose moiety to a molecule containing QA-tri(R / X)-FRX to form a molecule containing QA-tri(R / X)-FRXX. This step can be performed by the enzyme Qs-28-O-XylT4 of SEQ ID NO: 8 or a sequence having at least 70% sequence identity to SEQ ID NO: 8. Qs-28-O-XylT4 can be encoded by the polynucleotide sequence of SEQ ID NO: 7 or a sequence having at least 70% sequence identity to SEQ ID NO: 7.

[0087] This optional step also encompasses an enzyme having at least 70% sequence identity to the sequence of Qs-28-O-XylT4 (SEQ ID NO: 8). The amino acid sequence of the Qs-28-O-XylT4 enzyme may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8. Thus, in some embodiments, Qs-28-O-XylT4 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 8. For the enzymes defined herein with respect to sequence identity, typically, the function of adding a UDP-α-D-xylose moiety on a molecule containing QA-tri(R / X)-FRX to form a molecule containing QA-tri(R / X)-FRXX is retained.

[0088] F * Another optional step for forming F is to add a UDP-α-D-apiose moiety to the UDP-α-D-xylose moiety on a molecule containing QA-tri(R / X)-FRX to form a molecule containing QA-tri(R / X)-FRXA. This step can be carried out by the enzyme Qs-28-O-ApiT4 of SEQ ID NO: 10 or a sequence having at least 70% sequence identity to SEQ ID NO: 10. Qs-28-O-ApiT4 can be encoded by the polynucleotide sequence of SEQ ID NO: 9 or a sequence having at least 70% sequence identity to SEQ ID NO: 9.

[0089] This step also encompasses an enzyme having at least 70% sequence identity to the sequence of Qs-28-O-ApiT4 (SEQ ID NO: 10). The amino acid sequence of the Qs-28-O-ApiT4 enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10. Thus, in some embodiments, Qs-28-O-ApiT4 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 10. For the enzymes defined herein with respect to sequence identity, typically, they retain the function of adding a UDP-α-D-apiose moiety to the UDP-α-D-xylose moiety on a molecule containing QA-tri(R / X)-FRX to form a molecule containing QA-tri(R / X)-FRXA.

[0090] These steps form the F of the QA backbone * to form.

[0091] The method of the second form of the present invention is carried out in a biological system or a host. A polynucleotide encoding one or more of the above enzymes is introduced into the biological system and expressed. In most cases, since the biological system does not naturally express any of the enzymes of the second form of the present invention, the biological system can be engineered to express all the enzymes.

[0092] The biological system can be a plant or a microorganism. When the biological system is a plant, the plant can be a crop such as sunflower, potato, canola, dry bean, field pea, flax, safflower, buckwheat, cotton, corn, soybean, sugar beet, etc. The plant can also be corn, wheat, rapeseed and rice. Preferably, the plant is Nicotiana benthamiana.

[0093] In certain embodiments of the method of the second form of the present invention, the biological system is not Quillaja saponaria. When the biological system is a microorganism, the microorganism may be a bacterium or yeast.

[0094] Yeast (Saccharomyces cerevisiae) is a heterologous host used for the production of high-value low-molecular-weight compounds, including terpenoids. Similar to plants, yeast endogenously produces 2,3-oxidosqualene, which is a precursor of triterpenoids, and thus is a promising host for the industrial-scale production of triterpenoids. Yeast is also a very effective host for the functional expression of plant CYPs in the endoplasmic reticulum membrane. Modification of the triterpenoid skeleton by endogenous yeast enzymes is minimal, facilitating product purification. Yeast can be a production host that produces triterpenoids with diverse glycoside conjugates composed of multiple types of linear and branched saccharides. The glycosylation reaction in yeast is limited by the limited types of endogenous sugar donors. However, by expressing genes from higher plants, the nucleotide sugar metabolism of yeast can be expanded to include not only UDP-glucose and UDP-galactose but also UDP-rhamnose, UDP-glucuronic acid, UDP-xylose, UDP-arabinose, etc.

[0095] The method of the first aspect of the present invention may be carried out in vitro. "In vitro" means, in the context of the present invention, enzymatically treating an appropriate QA-tri(X / R)-F * derivative with an appropriate enzyme of the present invention. The QA-tri(X / R)-F * derivative may be either biosynthesized or chemically synthesized. The enzyme may be cloned or purified from a natural environment. Determining the optimal conditions for the enzyme treatment (e.g., time, temperature, buffer, etc.) is within the scope of those skilled in the art.

[0096] The identity of the QA derivative can be confirmed, for example, by elucidating its structure by NMR as described in "Materials and Methods".

[0097] In the second, third, and fourth embodiments of the present invention, the amino acid sequence of SEQ ID NO: 60 is encoded by the polynucleotide sequence of SEQ ID NO: 59; the amino acid sequence of SEQ ID NO: 58 is encoded by the polynucleotide sequence of SEQ ID NO: 57; and the amino acid sequence of SEQ ID NO: 56 is encoded by the polynucleotide sequence of SEQ ID NO: 55.

[0098] The methods of the second, third, and fourth embodiments of the present invention transform a host with a polynucleotide by introducing into a host cell via a vector a polynucleotide necessary for the biosynthesis of a molecule comprising QA-Tri(X / R)-F * -GR-Ac. Recombination can occur between the vector and the host cell genome, and the polynucleotide can be introduced into the host cell genome.

[0099] The fifth embodiment of the present invention is a glucosyltransferase enzyme of SEQ ID NO: 56 (QS-7-GlcT) or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO: 56. This enzyme can transfer a glucose residue to the C-3 position of the rhamnose residue of F * of the QS-7 precursor. This enzyme is as described in connection with the methods of the first to fourth embodiments of the present invention and has the same properties and functions as described in connection with the methods of the first to fourth embodiments of the present invention.

[0100] The glucosyltransferase enzyme can be encoded by the polynucleotide of SEQ ID NO: 55 or a polynucleotide molecule encoding an amino acid according to the fifth embodiment of the present invention. The QS-7-GlcT enzyme can be encoded, for example, by the polynucleotide sequence according to SEQ ID NO: 55 or by a degenerate code, or by a sequence that also encodes an enzyme according to the fifth embodiment of the present invention.

[0101] The fifth form of this invention includes a glucosyltransferase enzyme having at least 70% sequence identity to the sequence of QS-7-GlcT (SEQ ID NO: 56). The amino acid sequence of the QS-7-GlcT enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 56. Thus, in some embodiments, QS-7-GlcT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 56. For the enzymes defined herein with respect to sequence identity, typically, it adds a glucose moiety to a molecule containing QA-tri(X / R)-F * and retains the function of forming QA-tri(X / R)-F * G.

[0102] The sixth form of this invention is a rhamnosyltransferase enzyme of SEQ ID NO: 58 (QS-7-RhaT) or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO: 58. This enzyme can transfer a rhamnose moiety to the C-3 position of the D-fucose of F * of the QS-7 precursor. This enzyme is as described in the methods of the first to fourth forms of this invention and has the same properties and functions as described in relation to the methods of the first to fourth forms of this invention.

[0103] The rhamnosyltransferase enzyme can be encoded by the polynucleotide of SEQ ID NO: 57 or a polynucleotide molecule encoding an amino acid according to the sixth form of this invention. The QS-7-RhaT enzyme can be encoded, for example, by a polynucleotide sequence according to SEQ ID NO: 57 or by a degenerate code, or by a sequence encoding an enzyme according to the sixth form of this invention.

[0104] The sixth form of the present invention encompasses a rhamnosyltransferase enzyme having at least 70% sequence identity to the sequence of QS-7-RhaT (SEQ ID NO: 58). The amino acid sequence of the QS-7-RhaT enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 58. Thus, in some embodiments, QS-7-RhaT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 58. For the enzymes defined herein with respect to sequence identity, typically, the F of the QS-7 precursor * retains the function of transferring a rhamnose moiety to the C-3 position of D-fucose.

[0105] The seventh form of the present invention is an acetyltransferase enzyme of SEQ ID NO: 60 (QS-7-AcetylT) or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO: 60. This enzyme can transfer an acyl unit to the C-4 position of D-fucose of F of the QS-7 precursor. This enzyme is as described in the methods of the first to fourth forms of the present invention and has the same properties and functions as described in relation to the methods of the first to fourth forms of the present invention. * The acetyltransferase enzyme can be encoded by the polynucleotide of SEQ ID NO: 59 or a polynucleotide molecule encoding an amino acid according to the seventh form of the present invention. The QS-7-AcetylT enzyme can be encoded, for example, by a polynucleotide sequence according to SEQ ID NO: 59 or by a degenerate code, or by a sequence encoding an enzyme according to the seventh form of the present invention.

[0106]

[0107] ​The seventh form of the present invention includes an acetyltransferase enzyme having at least 70% sequence identity to the sequence of QS-7-AcetylT (SEQ ID NO: 60). The amino acid sequence of the QS-7-AcetylT enzyme can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 60. Thus, in some embodiments, QS-7-AcetylT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 60. For the enzymes defined herein with respect to sequence identity, typically, the F of the QS-7 precursor * retains the function of transferring an acyl unit to the C-4 position of the D-fucose.

[0108] Any percentage of sequence identity of the fifth, sixth and seventh forms of the present invention can be combined with any other percentage of sequence identity of the fifth, sixth and seventh forms of the present invention.

[0109] The eighth form of the present invention is a polynucleotide encoding one or more of the enzymes of the fifth to seventh forms of the present invention.

[0110] The ninth form of the present invention is a vector containing one or more of the polynucleotides according to the eighth form of the present invention.

[0111] The vector may contain one, two or three of the polynucleotides encoding the enzymes of the fifth to seventh forms of the present invention. Preferably, the vector contains three polynucleotides encoding the enzymes of the fifth to seventh forms of the present invention, or a corresponding number of vectors containing the three polynucleotides together.

[0112] The tenth form of the present invention is a host cell containing one or more of the polynucleotides according to the eighth form of the present invention.

[0113] The host cell may be a plant cell or a microbial cell. When the host cell is a microbial cell, it is preferably a yeast cell. When the host cell is a plant cell, it is preferably Nicotiana benthamiana.

[0114] A further feature of the tenth aspect of the present invention is a method for introducing the polynucleotide of the eighth aspect of the present invention into a host cell. The polynucleotide may be introduced into the host cell via a vector. Recombination may occur between the vector and the host cell genome, and the polynucleotide may be introduced into the host cell genome. Alternatively, the polynucleotide may be introduced into the host cell by co-invasion with a plurality of recombinant vectors. The recombinant vector may be an Agrobacterium tumefaciens strain described below.

[0115] The eleventh aspect of the present invention is a host cell transformed with the vector according to the ninth aspect of the present invention.

[0116] The twelfth aspect of the present invention is a plant or microbial biological system comprising the host cells defined in the tenth and eleventh aspects. This biological system may be a plant or a microorganism. When the biological system is a plant, it may be Nicotiana benthamiana or any of the plants described above. The method for producing a plant includes the steps of introducing the polynucleotide of the present invention into a host plant cell and regenerating a plant from the transformed host plant cell. When the biological system is a microorganism, it may be yeast.

[0117] The present invention also includes methods for producing each enzyme and each polynucleotide of the above aspects of the present invention, methods for producing a vector containing one or more of the polynucleotides of the present invention, host cells of the tenth and eleventh aspects of the present invention, and methods for producing the biological systems of the twelfth aspect of the present invention. These methods use techniques and materials well known in the art, such as WO2019 / 122259 and WO2020 / 260475, and are described in more detail as follows.

[0118] The polynucleotide of the present invention can be included in a vector, particularly an expression vector. The vector can be any double-stranded or single-stranded linear or circular plasmid, cosmid, phage, or Agrobacterium vector capable of transforming a prokaryotic or eukaryotic host by integration into the cell genome or other means. The vector can be an expression vector containing an inducible promoter and operably linked to the polynucleotide sequence. Typically, the vector may contain an enhancer sequence between the inducible promoter and the polynucleotide sequence. The vector may contain a terminator sequence and optionally a 3'UTR located upstream of the terminator sequence. The vector preferably may contain one or more polynucleotides encoding the enzymes of the fifth to seventh forms of the present invention, and preferably may contain all the sequences necessary to generate one version of the molecule defined according to the first and second forms of the present invention. The vector can be a plant vector or a microbial vector.

[0119] The polynucleotide in the vector is under the control of an appropriate promoter or other regulatory element for transcription in the host cell and may be operably linked to them. The host cell may be a yeast cell, a bacterial cell, or a plant cell. The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory elements. The advantage of using the native promoter is that it may be possible to avoid pleiotropic responses. In the case of cDNA, it may be under the control of an appropriate promoter or other regulatory element for expression in the host cell.

[0120] Preferred vectors for use in plants contain border sequences that allow the introduction and integration of the expression vector into the plant genome. The vector may be a plant binary vector.

[0121] The vector may be introduced into host cells in any biological system. The host may be a microorganism such as Escherichia coli or yeast. The vector may be part of Agrobacterium tumefaciens strain and may be used to infect a biological plant host system. Each of Agrobacterium tumefaciens contains one of the polynucleotides necessary to encode for the present invention and can be combined to co-infect host cells, whereby the host cells contain all the polynucleotides necessary to encode the enzymes of the fifth to seventh forms of the present invention.

[0122] The present invention also includes the step of culturing or growing a host for the production, harvesting and isolation of the desired QA-tri(X / R)-F * -GR-Ac derivative.

[0123] A further feature of the first to fourth forms of the present invention is the step of isolating the QA-tri(X / R)-F * -GR-Ac derivative.

[0124] The thirteenth form of the present invention is the QA-tri(X / R)-F obtained by the method of the present invention, particularly the method of the first to fourth forms of the present invention. * -GR-Ac derivative. The QA-tri(X / R)-F*-GR-Ac derivative obtained by the method of the present invention may be isolated from a biological system. The isolated QA-tri(X / R)-F * -GR-Ac derivative is QA-triR-FRXGR-Ac, QA-triR-FRXX-GR-Ac, QA-triR-FRXA-GR-Ac, QA-triX-FRXGR-Ac, QA-triX-FRXX-GR-Ac, QA-triX-FRXA-GR-Ac, QA-tri(X / R)-FRXGR-Ac, QA-tri(X / R)-FRXX-GR-Ac and / or QA-tri(X / R)-FRXA-GR-Ac or a mixture thereof. The QA-tri(X / R)-F * -GR-Ac derivative in this form of the present invention may be obtained by the method of the present invention. The QA-tri(X / R)-F *The -GR-Ac derivative may preferably be QA-triX-FRXA-GR-Ac.

[0125] A further form of the invention is a method for producing a QA-tri(X / R)-F-GR-Ac derivative, which includes the step of isolating a QA derivative and includes the steps of the method of the invention. * -GR-Ac derivative.

[0126] A fourteenth form of the invention is the use of a QA-tri(X / R)-F-GR-Ac derivative, particularly QA-triX-FRXA-GR-Ac, as an adjuvant in a vaccine composition after being isolated once from a biological system. The adjuvant may be a liposome formulation or an immunostimulating complex (ISCOM) formulation. * -GR-Ac derivative, particularly QA-triX-FRXA-GR-Ac, as an adjuvant in a vaccine composition after being isolated once from a biological system. The adjuvant may be a liposome formulation or an immunostimulating complex (ISCOM) formulation.

[0127] A further feature of the fourteenth form of the invention is that the adjuvant further contains a TLR4 agonist. The TLR4 agonist may be 3D-MPL. The QA-tri(X / R)-F-GR-Ac derivative of the invention may be combined with a further immunostimulant such as a TLR4 agonist, particularly a lipopolysaccharide TLR4 agonist such as a lipid A derivative, particularly monophosphoryl lipid A, for example 3-de-O-acylated monophosphoryl lipid A (3D-MPL). 3D-MPL is sold under the name "MPL" by GlaxoSmithKline Biologicals N.A. See, for example, U.S. Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL can be produced according to the method described in UK Patent No. 2220211A. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A and 4, 5 or 6 acyl chains. * -GR-Ac derivative may be combined with a further immunostimulant such as a TLR4 agonist, particularly a lipopolysaccharide TLR4 agonist such as a lipid A derivative, particularly monophosphoryl lipid A, for example 3-de-O-acylated monophosphoryl lipid A (3D-MPL). 3D-MPL is sold under the name "MPL" by GlaxoSmithKline Biologicals N.A. See, for example, U.S. Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL can be produced according to the method described in UK Patent No. 2220211A. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A and 4, 5 or 6 acyl chains.

[0128] Other TLR4 agonists that may be combined with the QA derivative of the invention include glucopyranosyl lipid adjuvants (GLA) as described in WO2008 / 153541, WO2009 / 143457 and the literature (Coler et al. 2011 and Arias et al. 2012).

[0129] A further feature of the fourteenth form of the present invention is that, for example, a QA-tri(X / R)-FRXA-GR-Ac derivative such as QA-tri(X / R)-F * The GR-Ac derivative is purified from the bark of Quillaja saponaria or produced by biosynthesis and combined with QS-21.

[0130] Also, the adjuvant of the present invention may be formulated in a suitable carrier such as an emulsifier (e.g., an oil-in-water emulsifier), liposomes, or an immunostimulating complex (ISCOM) as described below.

[0131] Liposomes The term liposome is well known in the art and defines a general category of vesicles containing a water space surrounded by one or more lipid bilayers. Liposomes consist of a bilayer of one or more lipids and / or phospholipids and may contain other molecules such as proteins and carbohydrates within their structure. Because both a lipid phase and an aqueous phase are present, liposomes can encapsulate or trap water-soluble substances, lipid-soluble substances, and / or amphiphilic compounds. Methods for the production of such liposomes are described in WO2013 / 041572.

[0132] The size of liposomes can vary from 30 nm to several μm depending on the lipid composition and the method used for their preparation.

[0133] The size of liposomes ranges from 50 nm to 200 nm, particularly in the range of 60 nm to 180 nm, for example, in the range of 70 to 165 nm. Optimally, the liposomes are stable and have a diameter of 100 nm to allow for simple sterilization by filtration.

[0134] The structural integrity of liposomes can be evaluated by methods such as dynamic light scattering (DLS) that measure the size (Z-average diameter, Zav) and polydispersity of liposomes, or by an electron microscope for the analysis of the liposome structure. The average particle size may be in the range of 95 to 120 nm, and / or the polydispersity (Pdl) index may be 0.3 or less (e.g., 0.2 or less).

[0135] ISCOM The term immunostimulating complex (ISCOM) is well known in the art and defines a delivery system for antigens and adjuvants within the same particle. ISCOMs are spherical, hollow, cage-like self-assembling particles.

[0136] Saponin-based adjuvants can be formulated in ISCOMs and / or ISCOM-matrix structures. ISCOMs can be prepared by the methods described in EP0109942B1, WO87 / 02250, and EP0180546B1. Transport and / or passenger antigens can be used by the methods described in WO9730728A1.

[0137] An ISCOM may be an ISCOM matrix complex containing at least one saponin fraction and a lipid. The lipid may be a sterol such as cholesterol. The ISCOM matrix complex may also contain a phospholipid such as phosphatidylcholine. The ISCOM matrix complex may contain one or more other immunomodulatory (adjuvant-active) substances and can be manufactured as described in EP0436620B1. The ISCOM matrix may be formulated as a mixture with an antigen, and the binding between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0138] An ISCOM may be an ISCOM complex containing at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex contains an antigen bound by surfactant treatment such that a portion of the antigen accumulates in the particles.

[0139] In some embodiments, the saponin fraction or at least one additional adjuvant is the QA derivative QA-tri(X / R)-F * -GR-Ac (e.g., QA-tri(X / R)-FRXA-GR-Ac), or QS-21 which is a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or is selected from any purified sub-fraction.

[0140] ISCOM particles may contain one or at least two saponin fractions. ISCOM particles may contain at least two saponin fractions of the same or different weight %. For example, the particles may be the QA derivative QA-tri(X / R)-F which is a QA derivative of any weight % * -GR-Ac and another saponin fraction such as QS-21 of any weight %. Thus, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1 to 99.9 wt%, 5 to 95 wt%, 10 to 90 wt%, 15 to 85 wt%, 20 to 80 wt%, 25 to 75 wt%, 30 to 70 wt%, 35 to 65 wt%, 40 to 60 wt%, 45 to 55 wt%, 40 to 60 wt%, or 5 wt% of one saponin fraction, for example the QA derivative QA-tri(X / R)-F * -GR-Ac, and the remainder may contain up to 100% of another saponin, for example QS-21. The weight is calculated as the total weight of the saponin fraction. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in US Application Publication No. 2013 / 0129770.

[0141] The ISCOM matrix or ISCOM complex is 5 to 99 wt% of one fraction, for example the QA derivative QA-tri(X / R)-F *-It may contain -GR-Ac, and the remainder may contain up to 100% by weight of another fraction, for example QS-21. The ISCOM matrix or ISCOM complex may contain at least two saponin fractions in the same or different weight percentages. The weight is calculated as the total weight of the saponin fractions. The ISCOM matrix or ISCOM complex is one fraction of 40% to 99% by weight, for example QA-tri(X / R)-F which is a QA derivative * -It may contain -GR-Ac and another fraction of 1% to 60% by weight, for example QS-21. The ISCOM matrix or ISCOM complex is one fraction of 70% to 95% by weight, for example QA-tri(X / R)-F which is a QA derivative * -It may contain -GR-Ac and another fraction of 30% to 5% by weight, for example QS-21.

[0142] ISCOM matrix particles and ISCOM complex particles may each be formed using only one type of saponin fraction. The composition may contain a plurality of particles, and each particle may contain only one type of saponin fraction. The composition may contain one or more different types of particles (for example, ISCOM-matrix complex particles, ISCOM complex particles), and each individual particle contains one type of saponin fraction. The saponin fraction in one particle may be different from the saponin fractions of other particles.

[0143] One type of saponin fraction or crude saponin fraction may be incorporated into one ISCOM matrix complex or particle, and another type of saponin fraction or crude saponin fraction may be incorporated into another ISCOM matrix complex or particle. The composition or vaccine may contain at least two types of complexes or particles, and each type contains one saponin incorporated into physically different particles.

[0144] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles in which two saponin fractions are individually incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles may be used.

[0145] The composition may comprise an ISCOM matrix or ISCOM complex particles, each having one saponin fraction. The composition may contain the particles in different or the same weight percentages. For example, the composition may comprise an ISCOM matrix or complex containing one first saponin fraction of 0.1-99.9 wt%, 5-95 wt%, 10-90 wt%, 15-85 wt%, 20-80 wt%, 25-75 wt%, 30-70 wt%, 35-65 wt%, 40-60 wt%, 45-55 wt%, 40-60 wt%, or 5 wt%, and the remaining portion composed of an ISCOM matrix or complex containing a different saponin fraction.

[0146] The saponin fraction in the first ISCOM matrix or ISCOM complex particles may be QA-tri(X / R)-F, a QA derivative * -GR-Ac, and the saponin fraction in the second ISCOM matrix or ISCOM complex particles may be QS-21.

[0147] A preferred composition comprises a first ISCOM matrix containing QA-tri(X / R)-F * -GR-Ac, a QA derivative, and a second ISCOM matrix containing QS-21, wherein the first ISCOM matrix constitutes about 70% of the weight of the total saponin adjuvant, and the second ISCOM matrix constitutes about 30% of the weight of the total saponin adjuvant. Another preferred composition comprises a first ISCOM matrix containing QA-tri(X / R)-F * -GR-Ac, a QA derivative, and a second ISCOM matrix containing QS-21, wherein the first ISCOM matrix constitutes about 85% of the weight of the total saponin adjuvant, and the second ISCOM matrix constitutes about 15% of the weight of the total saponin adjuvant. Thus, in a particular composition, the first ISCOM matrix is present in the range of about 70% to about 85% and the second ISCOM matrix is present in the range of about 15% to about 30% with respect to the total weight of the saponin adjuvant in the composition.

[0148] Saponin-based adjuvants may be Matrix-M TM as an adjuvant. Matrix-M TM adjuvant can be extracted from the bark of Quillaja saponaria Molina. This adjuvant can be formulated and purified together with cholesterol and phospholipids. Matrix-M TM The adjuvant may consist of two separately formed groups of particles having complementary properties. These particles may be about 25 - 55 nm, about 30 - 50 nm, or about 35 - 45 nm, and preferably, the particles are 40 nm.

[0149] Matrix-M TM One particle of Matrix-M may be QA - tri(X / R)-F * -GR-Ac (particle 1), and the other particle may be QS-21 (particle 2). Matrix-M TM Matrix-M may contain the two particles in a ratio necessary to maintain high adjuvant activity and an optimal safety margin. For example, Matrix-M TM contains 85% particle 1 and 15% particle 2. Matrix-M TM contains 92% particle 1 and 8% particle 2.

[0150] Matrix-M TM The dosage of the adjuvant is about 1 - about 100 μg, about 5 - about 95 μg, about 10 - about 90 μg, about 15 - about 85 μg, about 20 - about 80 μg, about 25 - about 75 μg, about 30 - about 70 μg, about 35 - about 65 μg, about 40 - about 60 μg, about 45 - about 55 μg, about 50 μg, or any value in between.

[0151] Matrix-M TM The adjuvant can induce high levels of broadly reactive antibodies with a balanced TH1 and TH2 type response, including biologically active antibody isotypes such as mouse IgG2a, multifunctional T cells, and cytotoxic T lymphocytes, for a long period. Generally, Matrix-M TMAdjuvants can enhance the immune response and create an environment of activated cells including T cells, B cells, natural killer cells, neutrophils, monocytes, dendritic cells ww, resulting in a rapid and significant effect on the efflux of cells to the local lymph nodes. Matrix-M TM can enhance the combination of antibody and cellular immune responses.

[0152] The fifteenth form of the present invention is an adjuvant composition comprising a QA-tri(X / R)-F * -GR-Ac derivative or QA-triX-FRXA-GR-Ac according to the thirteenth form of the present invention.

Examples

[0153] The present invention will be described with reference to the following non-limiting examples:

[0154] Example 1 - Identification of QS-7-GlcT Previously, genomic and transcriptomic sequence resources of Q. saponaria were generated and used for the identification of genes required for the production of the saponin QA-tri(X / R)-FRX(A / X) (Figure 4). This saponin is a common precursor of various immunostimulatory saponins produced by Q. saponaria including QS-21 and QS-7. These sequence resources have revealed that the genes required for the biosynthesis of this backbone are co-expressed among different tissues of Q. saponaria and highly expressed in leaf primordia.

[0155] UDP-dependent glycosyltransferases (UGTs) are generally involved in the glycosylation of plant natural products (Louveau & Osbourn, 2019), and several enzymes are known to be required for the production of QA-tri(X / R)-FRX(A / X). Using the above sequence resources, one UGT was identified (QsUGT-BI) that showed an expression pattern similar to that of previously characterized enzymes. When QsUGT-BI was transiently expressed together with the Q. saponaria-derived genes required for the biosynthesis of the QA-tri X-FRXA backbone (Table 2), a new product with a mass suggestive of the addition of a hexose residue was identified by LC-MS analysis (Figure 5). Some characterized saponins from Q. saponaria are known to have a glucose residue added to the C-28 sugar chain (Fleck et al., 2019), and it is suggested that the hexose added by QsUGT-BI is probably glucose. One such saponin is QS-7, which is characterized by D-glucose added to the C-3 position of the rhamnose residue at C-28 (Figure 1). The product presumably assigned as QA-tri X-FRXA glucoside (QA-tri X-FRXA-G) was thought to be a precursor of QS-7. The putative glycosyltransferase QsUGT-BI is also referred to herein as QS-7-GlcT.

[0156] Example 2 - Identification of QS-7-AcetylT QS-7 is characterized by an acetyl group attached to the C-4 position of D-fucose (Figure 1). BAHD acyltransferases are generally known to be involved in the acylation of various plant specialized metabolites. As a result, a series of BAHD acyltransferases (ACTs) showing co-expression with known QA-triX-FRXA-G pathway genes were cloned and tested in N. benthamiana by co-injecting ACT candidates together with the genes necessary for the biosynthesis of the QA-triX-FRXA backbone. LC-MS analysis of leaf extracts revealed the detection of a new product in samples expressing the candidate "QsACT-19". This product was found to have the mass of QA-triX-FRXA with an acetyl group added (QA-triX-FRXA-Ac), indicating that QsACT-19’ is an acetyltransferase (Figure 6). Therefore, the putative QA-triX-FRXA-Ac product was assumed to be the QS-7 precursor. QsACT-19’ is also referred to herein as QS-7-AcetylT.

[0157] Example 3 - Identification of QS-7-RhaT Analysis of known saponins from Q. saponaria revealed that the presence of an additional rhamnose (attached to the C-3 position of D-fucose) in QS-7 is dependent on the presence of an acetyl group added to C-4 of D-fucose. As described above, the product of the QsACT-19’ enzyme (QA-triX-FRXA-Ac), which is characterized by an acetyl group at the same position as QS-7, was used as a scaffold to screen for more UGTs by transient expression. From among those candidates, one enzyme (QsUGT-0023500) resulted in the appearance of a new peak consistent with the addition of a deoxyhexose such as rhamnose to the QA-triX-FRXA-Ac product (Figure 7). The resulting product was assigned as QA-triX-FRXA-Ac rhamnoside (QA-triX-FRXA-R-Ac). QsUGT-0023500 is also referred to herein as Qs-7-RhaT.

[0158] Generation of Example 4-QS-7 (QA-triX-FRXA-GR-Ac) Following the identification of QsUGT-BI, QsUGT-0023500, and QsACT-19’, a series of transient expressions were carried out by combining the three newly identified genes with the constructs required for the production of QA-triX-FRXA. LC-MS analysis of the leaf extracts revealed a peak that matched the retention time and mass spectrum of the QS-7 standard, a fraction purified from the bark of Q. saponaria (Figure 8). This peak was not observed in control samples lacking any one of the three newly identified enzymes. This would seem to support the involvement of these enzymes in QS-7 production. Nevertheless, to determine clear QS-7 production, large-scale infiltration was carried out using a total of 410 plants. Extraction of the leaf material enabled the isolation of the new product by preparative HPLC. The identity of the new product was 1 confirmed by 1H NMR analysis (Figures 9 and 10).

[0159] Materials and Methods Primers and Cloning The genes encoding the enzymes described herein (QS-7-RhaT / QsUGT-0023500, QS-7-GlcT / QsUGT-BI and QsAcetylT / QsACT-19’) were amplified by PCR from cDNA derived from leaf tissue of Q. saponaria. PCR was carried out using the primers detailed in Table 1 and iProof polymerase and thermal cycling according to the manufacturer's recommendations. The resulting PCR products were purified (Qiagen PCR cleanup kit) and each was cloned into the pDONR207 vector using BP Clonase according to the manufacturer's instructions. The BP reaction was transformed into E. coli, the resulting transformants were cultured and the plasmids were isolated by miniprep (Qiagen). The isolated plasmids were sequenced (Eurofins) to confirm the presence of the correct genes. Next, each of the three genes was further subcloned into the pEAQ-HT-DEST1 expression vector using LR Clonase. A. tumefaciens LBA4404 was transformed using the resulting vectors by rapid freezing in liquid nitrogen.

[0160]

Table 1

[0161] Agroinfiltration of N. benthamiana leaves As previously described (Reed et al., 2017), agroinfiltration was performed using a needleless syringe. As described above, all genes were expressed from the pEAQ-HT-DEST1 binary expression vector (Sainsbury et al., 2009) in A. tumefaciens LBA4404. In some cases, multiple genes were integrated into a single Golden Gate binary vector to facilitate infiltration. Bacterial and plant cultures were as described in (Reed et al., 2017).

[0162] Preparation of N. benthamiana leaf extract for LC-MS analysis Leaves were harvested 5 days after agroinfiltration and freeze-dried. The dried leaf material (10 mg per sample) was ground at 1000 rpm for 1 minute using tungsten beads (Geno / Grinder 2010, Spex SamplePrep). Metabolites were extracted with 550 μL of 80% methanol containing 20 μg / mL of internal standard (digitoxin (Sigma-Aldrich)) and incubated at 18 °C for 20 minutes while shaking at 1400 rpm (Thermomixer Comfort, Eppendorf). Each sample was defatted by partitioning twice with 400 μL of hexane. The upper phase was discarded, and the lower aqueous phase was vacuum-dried at 40 °C for 1 hour (EZ-2 Series Evaporator, Genevac). The dried material was resuspended in 75 μL of 100% methanol, centrifuged at 12,500 x g for 30 seconds (0.2 μm, Spin-X, Costar), and filtered. The filtrate (50 μL) was mixed with 50 μL of 50% methanol in a glass vial and analyzed as detailed below.

[0163] HPLC-CAD-MS analysis of N. benthamiana leaf extract Sample analysis was performed using a QExactive mass spectrometer (Thermo Fisher) equipped with a 50 x 2.1 mm 2.6 μ Kinetix XB-C18 column (Phenomenex). The solvent system consisted of water [A] containing 0.1% formic acid and acetonitrile [B], and the flow rate was 0.6 mL / min. The program consisted of 15% [B] for 0.75 minutes, followed by a stepwise increase to 60% [B] until 13 minutes. The percentage of [B] was increased to 100% in 0.25 minutes, maintained for 1 minute, then returned to 15% in 0.25 seconds and maintained for 2 minutes. CAD and MS were set to negative mode, and the samples were monitored in the range of 400 - 2500 m / z.

[0164] Large-scale vacuum infiltration of N. benthamiana As previously described (Reed et al., 2017; Stephenson et al., 2018), plants were vacuum infiltrated using the A. tumefaciens LBA4404 strain carrying the pEAQ-HT-DEST1 expression vector harboring the relevant genes detailed in Table 2 (Reed et al., 2017; Stephenson et al., 2018).

[0165] Purification of QS-7 by large-scale infiltration of N. benthamiana A series of A. tumefaciens cultures containing the constructs related to QS-7 production were co-infiltrated into N. benthamiana by large-scale vacuum infiltration. A total of 410 plants were agro-infiltrated, and the leaves were harvested 5 days later and freeze-dried to obtain 104 g of dry matter. The leaf material was first defatted with hexane and then thoroughly extracted using methanol. The methanol extracts were combined and evaporated under reduced pressure. Before partitioning in a separatory funnel using a mixed solvent of hexane, dichloromethane, ethyl acetate and n-butanol, the dry extract was dissolved in the minimum amount of methanol and diluted with an equal volume of water. The butanol layer was recovered, dried over anhydrous sodium sulfate, evaporated under reduced pressure and redissolved in the minimum amount of methanol. Purification of QS-7 was carried out by reverse-phase semi-preparative HPLC using a Luna C18 column 250 x 10 mm (particle size 5 μm) (Phenomenex). The mobile phase consisted of a weak solvent [A] (20 mM NH4HCO3 (pH 8.6)) and a strong solvent acetonitrile [B]. The separation program was maintained at 25% [B] for the first 2 minutes, then a gradient of 25 - 70% for 18 minutes and 70% for 5 minutes. The column was equilibrated with 25% [B] for 2 minutes between analyses. As a result, 1 A pure semi-purified fraction (about 12 mg of light brown amorphous substance) containing 3 - 5% of QS-7 was obtained by 1H NMR analysis.

[0166] NMR analysis 1D and 2D NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer (JIC, UK) equipped with a BBFO Plus Smart probe and a triple resonance TCI cryoprobe. Chemical shifts are relative to the residual signal of the solvent (MeOH-d4; δH 3.31; δC 49.15).

[0167]

Table 2

[0168] Clauses Embodiments of the invention are described in the claims and the following clauses.

[0169] 1. A method for producing biosynthetic QA-tri(X / R)-F * -GR-Ac in a host, the method comprising the following steps: in the host a) expressing the genes necessary for the biosynthesis of QA-tri R-F * and / or QA-tri X-F * and b) introducing a polynucleotide encoding: i. an enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. an enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, and iii. an enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58.

[0170] 2. The method according to clause 1, wherein tri(X / R) is tri X and F * is FRXA.

[0171] 3. Step a) is: into a host 1) expressing in the host the genes necessary for the biosynthesis of QA-tri R and / or QA-tri X, and 2) introducing a polynucleotide encoding: i. the enzyme of kiy acid 28-O-fucosyltransferase (Qs-28-O-FucT, SEQ ID NO: 2) or a sequence having at least 70% sequence identity to SEQ ID NO: 2, optionally, the enzyme from Q. saponaria (QsFucSyn, SEQ ID NO: 12) or a sequence having at least 45% sequence identity to SEQ ID NO: 12 that increases the production of fucosylated saponin; ii. the enzyme of kiy acid 28-O-fucoside [1,2]-rhamnosyltransferase (Qs-28-O-RhaT, SEQ ID NO: 4) or a sequence having at least 70% sequence identity to SEQ ID NO: 4; iii. the enzyme of kiy acid 28-O-fucoside [1,2]-rhamnoside [1,4]xylosyltransferase (Qs-28-O-XylT3, SEQ ID NO: 6) or a sequence having at least 70% sequence identity to SEQ ID NO: 6; and iv. optionally, the enzyme of kiy acid 28-O-fucoside [1,2]-rhamnoside [1,4]xyloside [1,3]xylosyltransferase (Qs-28-O-XylT4, SEQ ID NO: 8) or a sequence having at least 70% sequence identity to SEQ ID NO: 8 and / or the enzyme of kiy acid 28-O-fucoside [1,2]-rhamnoside [1,4]xyloside [1,3]apiosyltransferase (Qs-28-O-ApiT4, (SEQ ID NO: 10) or a sequence having at least 70% sequence identity to SEQ ID NO: 10 The method according to item 1, comprising.

[0172] 4. Step a) is: into a host 1) expressing in the host the genes necessary for the biosynthesis of QA-tri R and / or QA-tri X, and 2) introducing a polynucleotide encoding: i. An enzyme of Qs-28-O-FucT (SEQ ID NO: 2) or a sequence having at least 70% sequence identity, optionally an enzyme of QsFucSyn (SEQ ID NO: 12) or a sequence having at least 45% sequence identity; ii. An enzyme of Qs-28-O-RhaT (SEQ ID NO: 4) or a sequence having at least 70% sequence identity; iii. An enzyme of Qs-28-O-XylT3 (SEQ ID NO: 6) or a sequence having at least 70% sequence identity; and iv Optionally, an enzyme of Qs-28-O-ApiT4 (SEQ ID NO: 10) or a sequence having at least 70% sequence identity to SEQ ID NO: 10 The method according to claim 1, comprising.

[0173] 5. Step a) is: In the host 1) Expressing in the host the genes necessary for the biosynthesis of QA-triR and / or QA-triX, and 2) Introducing a polynucleotide encoding: i. An enzyme of Qs-28-O-FucT (SEQ ID NO: 2) or a sequence having at least 70% sequence identity, optionally an enzyme of QsFucSyn (SEQ ID NO: 12) or a sequence having at least 45% sequence identity; ii. An enzyme of Qs-28-O-RhaT (SEQ ID NO: 4) or a sequence having at least 70% sequence identity; iii. An enzyme of Qs-28-O-XylT3 (SEQ ID NO: 6) or a sequence having at least 70% sequence identity; and iv Optionally, an enzyme of Qs-28-O-XylT4 (SEQ ID NO: 8) or a sequence having at least 70% sequence identity to SEQ ID NO: 8 The method according to claim 1, comprising.

[0174] 6. Step a) is: In the host 1) Expressing in the host the genes necessary for the biosynthesis of QA-triX, and 2) Introducing a polynucleotide encoding: i. An enzyme of Qs-28-O-FucT (SEQ ID NO: 2) or a sequence having at least 70% sequence identity, optionally an enzyme of QsFucSyn (SEQ ID NO: 12) or a sequence having at least 45% sequence identity; ii. An enzyme of Qs-28-O-RhaT (SEQ ID NO: 4) or a sequence having at least 70% sequence identity; iii. An enzyme of Qs-28-O-XylT3 (SEQ ID NO: 6) or a sequence having at least 70% sequence identity; and iv. An enzyme of Qs-28-O-ApiT4 (SEQ ID NO: 10) or a sequence having at least 70% sequence identity to SEQ ID NO: 10 The method according to claim 2, comprising the above.

[0175] 7. Step 1) is a polynucleotide encoding the following: i. Kiraic acid 3-O-glucuronosyltransferase (QsCSL1, SEQ ID NO: 26) or kiraic acid 3-O-glucuronosyltransferase glucuronosyltransferase (QsCslG2, SEQ ID NO: 28), or an enzyme of a sequence having at least 70% sequence identity to SEQ ID NO: 26 or 28; ii. Q. saponaria QA-mono-β-1,2-D-galactosyltransferase (Qs-3-O-GalT, SEQ ID NO: 30) or an enzyme of a sequence having at least 70% sequence identity; and iii. Q. saponaria QA-dialpha-1,3-L-rhamnosyltransferase (DN20529_c0_g2_i8, SEQ ID NO: 36), Q. saponaria QA-dialpha-1,3-L-rhamnosyltransferase (Qs_0283850, SEQ ID NO: 34), or Q. saponaria QA-didual beta-1,3-D-xylosyltransferase / alpha-1,3-L-rhamnosyltransferase (Qs-3-O-RhaT / XylT, SEQ ID NO: 32) or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO: 36, 34, or 32, and / or Q. saponaria QA-dibeta-1,3-D-xylosyltransferase (Qs_0283870, SEQ ID NO: 38) or Qs-3-O-RhaT / XylT (SEQ ID NO: 32) or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO: 38 or 32; introducing into a host A method according to any one of items 3 to 5, comprising

[0176] 8. Step 1) comprises a polynucleotide encoding the following: i. QsCSL1 (SEQ ID NO: 26) or QsCslG2 (SEQ ID NO: 28), or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO: 26 or 28; ii. Qs-3-O-GalT (SEQ ID NO: 30) or an enzyme having a sequence with at least 70% sequence identity; and iii. DN20529_c0_g2_i8 (SEQ ID NO: 36), Qs_0283850 (SEQ ID NO: 34), or Qs-3-O-RhaT / XylT (SEQ ID NO: 32) or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO: 36, 34, or 32 introducing into a host A method according to any one of items 3 to 5, comprising

[0177] 9. Step 1) comprises a polynucleotide encoding the following: i. An enzyme of QsCSL1 (SEQ ID NO: 26) or QsCslG2 (SEQ ID NO: 28), or a sequence having at least 70% sequence identity to SEQ ID NO: 26 or 28; ii. An enzyme of Qs-3-O-GalT (SEQ ID NO: 30) or a sequence having at least 70% sequence identity thereto; and iii. An enzyme of Qs_0283870 (SEQ ID NO: 38) or Qs-3-O-RhaT / XylT (SEQ ID NO: 32) or a sequence having at least 70% sequence identity to SEQ ID NO: 38 or 32 Introducing into a host The method according to any one of items 2 to 6, further comprising the above.

[0178] 10. Step a)-1) is a polynucleotide encoding the following: i. An enzyme of Q. Saponaria β-amylin synthase (QsbAS, SEQ ID NO: 18) or a sequence having at least 50% sequence identity to SEQ ID NO: 18; ii. An enzyme of Q. Saponaria kaurenoic acid C-28 oxidase (QsCYP716-C-28, SEQ ID NO: 20), or a sequence having at least 50% sequence identity to SEQ ID NO: 20; iii. An enzyme of Q. Saponaria kaurenoic acid C-16α oxidase (QsCYP716-C-16α, SEQ ID NO: 22), or a sequence having at least 50% sequence identity to SEQ ID NO: 22; and iv. An enzyme of Q. Saponaria kaurenoic acid C-23 oxidase (QsCYP714-C-23, SEQ ID NO: 24), or a sequence having at least 50% sequence identity to SEQ ID NO: 24; Introducing into a host The method according to any one of items 2 to 9, further comprising the above.

[0179] 11. Amino acid sequence number 2 is encoded by polynucleotide sequence number 1; Amino acid sequence number 4 is encoded by polynucleotide sequence number 3; Amino acid sequence number 6 is encoded by polynucleotide sequence number 5; Amino acid sequence number 8 is encoded by polynucleotide sequence number 7; Amino acid sequence number 10 is encoded by polynucleotide sequence number 9, The method according to any one of items 2 to 10.

[0180] 12. Amino acid sequence number 26 is encoded by polynucleotide sequence number 25; Amino acid sequence number 28 is encoded by polynucleotide sequence number 27; Amino acid sequence number 30 is encoded by polynucleotide sequence number 29; Amino acid sequence number 32 is encoded by polynucleotide sequence number 31; Amino acid sequence number 34 is encoded by polynucleotide sequence number 33; Amino acid sequence number 36 is encoded by polynucleotide sequence number 35; Amino acid sequence number 38 is encoded by polynucleotide sequence number 37; The method according to any one of items 5 to 11.

[0181] 13. QA-Tri(X / R)-F * -GR-Ac, wherein the acetyl (Ac) moiety is attached to the C-4 position of F * of D-fucose, the rhamnose (R) residue is attached to the C-3 position of F * of D-fucose, and the glucose (G) residue is attached to the C-3 position of the rhamnose residue of F * and QA-Tri(X / R)-F * is i. Enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. An enzyme having the amino acid sequence of SEQ ID NO: 60, namely QS-7-AcetylT, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 60; an enzyme having the amino acid sequence of SEQ ID NO: 62, namely SOAP10, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 62, or an enzyme having the amino acid sequence of SEQ ID NO: 64, namely DMOT9, or an enzyme having an amino acid sequence with at least 25% sequence identity to SEQ ID NO: 64, wherein one or more enzymes are selected, and iii. An enzyme having the amino acid sequence of SEQ ID NO: 58, namely QS-7-RhaT, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 58, in combination with QA-tri(X / R)-F * -GR-Ac to form a method comprising.

[0182] 14. i. Qs-28-O-FucT (SEQ ID NO: 2) or an enzyme of a sequence having at least 70% sequence identity, optionally QsFucSyn (SEQ ID NO: 12) or an enzyme of a sequence having at least 45% sequence identity to SEQ ID NO: 12; ii. Qs-28-O-RhaT (SEQ ID NO: 4) or an enzyme of a sequence having at least 70% sequence identity to SEQ ID NO: 4; iii. Qs-28-O-XylT3 (SEQ ID NO: 6) or an enzyme of a sequence having at least 70% sequence identity to SEQ ID NO: 6; and iv. Optionally, Qs-28-O-XylT4 (SEQ ID NO: 8) or an enzyme of a sequence having at least 70% sequence identity to SEQ ID NO: 8 and / or Qs-28-O-ApiT4 (SEQ ID NO: 10) or an enzyme of a sequence having at least 70% sequence identity to SEQ ID NO: 10: The method according to item 13, further comprising combining with.

[0183] 15. i. An enzyme of the sequence of Qs-28-O-FucT (SEQ ID NO: 2) or a sequence having at least 70% sequence identity to SEQ ID NO: 2, optionally, an enzyme of the sequence of QsFucSyn (SEQ ID NO: 12) or a sequence having at least 45% sequence identity to SEQ ID NO: 12; ii. An enzyme of the sequence of Qs-28-O-RhaT (SEQ ID NO: 4) or a sequence having at least 70% sequence identity to SEQ ID NO: 4; iii. An enzyme of the sequence of Qs-28-O-XylT3 (SEQ ID NO: 6) or a sequence having at least 70% sequence identity to SEQ ID NO: 6; and iv. Optionally, an enzyme of the sequence of Qs-28-O-ApiT4 (SEQ ID NO: 10) or a sequence having at least 70% sequence identity to SEQ ID NO: 10: The method according to item 13, further comprising combining with.

[0184] 16. i. An enzyme of the sequence of Qs-28-O-FucT (SEQ ID NO: 2) or a sequence having at least 70% sequence identity, optionally, an enzyme of the sequence of QsFucSyn (SEQ ID NO: 12) or a sequence having at least 45% sequence identity to SEQ ID NO: 12; ii. An enzyme of the sequence of Qs-28-O-RhaT (SEQ ID NO: 4) or a sequence having at least 70% sequence identity to SEQ ID NO: 4; iii. An enzyme of the sequence of Qs-28-O-XylT3 (SEQ ID NO: 6) or a sequence having at least 70% sequence identity to SEQ ID NO: 6; and iv. Optionally, an enzyme of the sequence of Qs-28-O-XylT4 (SEQ ID NO: 8) or a sequence having at least 70% sequence identity to SEQ ID NO: 8: The method according to item 13, further comprising combining with.

[0185] 17. The method according to item 13, wherein tri(X / R) is triX and F * is FRXA.

[0186] 18. i. An enzyme of a sequence having at least 70% sequence identity to Qs-28-O-FucT (SEQ ID NO: 2) or SEQ ID NO: 2, optionally, an enzyme of a sequence having at least 45% sequence identity to QsFucSyn (SEQ ID NO: 12) or SEQ ID NO: 12; ii. An enzyme of a sequence having at least 70% sequence identity to Qs-28-O-RhaT (SEQ ID NO: 4) or SEQ ID NO: 4; iii. An enzyme of a sequence having at least 70% sequence identity to Qs-28-O-XylT3 (SEQ ID NO: 6) or SEQ ID NO: 6; and iv. An enzyme of a sequence having at least 70% sequence identity to Qs-28-O-ApiT4 (SEQ ID NO: 10) or SEQ ID NO: 10: The method according to item 17, further comprising combining with.

[0187] 19. i. QsCSL1 (SEQ ID NO: 26) or QsCslG2 (SEQ ID NO: 28), or an enzyme of a sequence having at least 70% sequence identity to SEQ ID NO: 26 or 28; ii. An enzyme of a sequence having at least 70% sequence identity to Qs-3-O-GalT (SEQ ID NO: 30) or SEQ ID NO: 30; and iii. DN20529_c0_g2_i8 (SEQ ID NO: 36), Qs_0283850 (SEQ ID NO: 34), or an enzyme of a sequence having at least 70% sequence identity to Qs-3-O-RhaT / XylT (SEQ ID NO: 32) or SEQ ID NO: 36, 34 or 32, and / or an enzyme of a sequence having at least 70% sequence identity to Qs_0283870 (SEQ ID NO: 38) or Qs-3-O-RhaT / XylT (SEQ ID NO: 32) or SEQ ID NO: 38 or 32: The method according to any one of items 13 to 16, further comprising combining with.

[0188] 20. i. QsCSL1 (SEQ ID NO: 26) or QsCslG2 (SEQ ID NO: 28), or an enzyme of a sequence having at least 70% sequence identity to SEQ ID NO: 26 or 28; ii. an enzyme of Qs-3-O-GalT (SEQ ID NO: 30) or a sequence having at least 70% sequence identity to SEQ ID NO: 30; and iii. an enzyme of DN20529_c0_g2_i8 (SEQ ID NO: 36), Qs_0283850 (SEQ ID NO: 34), or Qs-3-O-RhaT / XylT (SEQ ID NO: 32) or a sequence having at least 70% sequence identity to SEQ ID NO: 36, 34, or 32: The method according to any one of items 13 to 16, further comprising combining with.

[0189] 21. i. an enzyme of QsCSL1 (SEQ ID NO: 26) or QsCslG2 (SEQ ID NO: 28), or a sequence having at least 70% sequence identity to SEQ ID NO: 26 or 28; ii. an enzyme of Qs-3-O-GalT (SEQ ID NO: 30) or a sequence having at least 70% sequence identity to SEQ ID NO: 30; and / or iii. an enzyme of Qs_0283870 (SEQ ID NO: 38) or Qs-3-O-RhaT / XylT (SEQ ID NO: 32) or a sequence having at least 70% sequence identity to SEQ ID NO: 38 or 32: The method according to any one of items 13 to 18, further comprising combining with.

[0190] 22. i. an enzyme of QsbAS (SEQ ID NO: 18) having at least 50% sequence identity to SEQ ID NO: 18; ii. an enzyme of QsCYP716-C-28 (SEQ ID NO: 20) having at least 50% sequence identity to SEQ ID NO: 20; iii. an enzyme of QsCYP716-C-16α (SEQ ID NO: 22) having at least 50% sequence identity to SEQ ID NO: 22, and iv. an enzyme of QsCYP714-C-23 (SEQ ID NO: 24) having at least 50% sequence identity to SEQ ID NO: 24: The method according to any one of items 13 to 21, further comprising combining with.

[0191] 23. QA-tri(X / R)-F * - The method according to any one of the preceding claims, further comprising the step of isolating the -GR-Ac derivative.

[0192] 24. QA-tri(X / R)-F * - The method according to claim 23, wherein the -GR-Ac is QA-triR-FRXGR-Ac, QA-triR-FRXX-GR-Ac, QA-triR-FRXA-GR-Ac, QA-triX-FRXGR-Ac, QA-triX-FRXX-GR-Ac, QA-triX-FRXA-GR-Ac, QA-tri(X / R)-FRXGR-Ac, QA-tri(X / R)-FRXX-GR-Ac and / or QA-tri(X / R)-FRXA-GR-Ac or a mixture thereof.

[0193] 25. QA-tri(X / R)-F * - The method according to claim 24, wherein the -GR-Ac is QA-triX-FRXA-GR-Ac.

[0194] 26. QA-tri(X / R)-F obtained by the method according to claim 23 * -GR-Ac.

[0195] 27. QA-tri(X / R)-F * - The method according to claim 26, wherein the -GR-Ac is QA-triR-FRXGR-Ac, QA-triR-FRXX-GR-Ac, QA-triR-FRXA-GR-Ac, QA-triX-FRXGR-Ac, QA-triX-FRXX-GR-Ac, QA-triX-FRXA-GR-Ac, QA-tri(X / R)-FRXGR-Ac, QA-tri(X / R)-FRXX-GR-Ac and / or QA-tri(X / R)-FRXA-GR-Ac, or a mixture thereof.

[0196] 28. QA-tri(X / R)-F * - The method according to claim 27, wherein the -GR-Ac is QA-triX-FRXA-GR-Ac.

[0197] Abbreviations Apif - D-apiose DMOT9 - (3S,5S,6S)-3,5-dihydroxy-6-methyloctanoyl-CoA transferase 9 DN20529_c0_g2_i8 - Q. saponaria QA-di-α-1,3-L-rhamnosyltransferase FR - disaccharide of β-D-fucose (F) and α-L-rhamnose (R) residues FRX - trisaccharide of β-D-fucose (F), α-L-rhamnose (R) and β-D-xylose (X) residues FRXX - trisaccharide of β-D-fucose (F), α-L-rhamnose (R), and two β-D-xylose (X, X) residues FRXA - tetrasaccharide of β-D-fucose (F), α-L-rhamnose (R), β-D-xylose (X) and β-D-apiose (A) residues FRXX / A - tetrasaccharide of FRXX or FRXA Fucp - D-fucopyranose FucSyn - enzyme that increases the production of fucosylated saponin FSL - QsFucSyn-Like Galp - D-galactopyranose GlcpA - D-glucopyranuronic acid Glcp - D-glucopyranose OS - 2,3-oxidosqualene OSC - oxidosqualene cyclase QA - quinic acid QA derivative - QA-di - 3-O-{β-D-galactopyranosyl-(1->2)-β-D-glucopyranosiduronic acid}-quinic acid - QA-di-F - 3-O-{β-D-galactopyranosyl-(1->2)-β-D-glucopyranosiduronic acid}-28-O-{β-D-fucopyranosyl ester}-quinic acid - QA-di-FR - 3-O-{β-D-galactopyranosyl-(1->2)-β-D-glucopyranuronate}-28-O-{α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-di-FRX - 3-O-{β-D-galactopyranosyl-(1->2)-β-D-glucopyranuronate}-28-O-{β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-di-FRXA - 3-O-{β-D-galactopyranosyl-(1->2)-β-D-glucopyranuronate}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-di-FRXX - 3-O-{β-D-galactopyranosyl-(1->2)-β-D-glucopyranuronate}-28-O-{β-D-xylopyranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-mono - 3-O-{β-D-glucopyranuronate}-oleanolic acid - QA-mono-F - 3-O-{β-D-glucopyranuronate}-28-O-{β-D-fucopyranosyl ester}-oleanolic acid - QA-mono-FR - 3-O-{β-D-glucopyranuronate}-28-O-{α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-mono-FRX - 3-O-{β-D-glucopyranuronate}-28-O-{β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-Mono-FRXA - 3-O-{β-D-glucopyranuronosyluronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-Mono-FRXX - 3-O-{β-D-glucopyranuronosyluronic acid}-28-O-{β-D-xylopyranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-TriR - 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronosyluronic acid}-oleanolic acid - QA-TriR-F - 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronosyluronic acid}-28-O-{β-D-fucopyranosyl ester}-oleanolic acid - QA-TriR-FR - 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronosyluronic acid}-28-O-{α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-TriR-FRX - 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronosyluronic acid}-28-O-{β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-TriR-FRXA - 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronosyluronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-oleanolic acid - QA-Tri R-FRXX - 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-xylopyranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-kic acid - QA-Tri X - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-kic acid - QA-Tri X-F - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-fucopyranosyl ester}-kic acid - QA-Tri X-FR - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-kic acid - QA-Tri X-FRX - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-kic acid - QA-Tri X-FRXA - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-kic acid - QA-Trix-FRXX - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-xylopyranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-cholic acid - QA-Trix-G - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-glucopyranosyl ester}-cholic acid - QA-Trix-GR - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{α-L-rhamnopyranosyl-(1->2)-β-D-glucopyranosyl ester}-cholic acid - QA-Trix-GRX - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-glucopyranosyl ester}-cholic acid - QA-Tri(X / R) - QA-Trix or QA-TriR, QA glycosylated at C-3 position with any of the branched-chain trisaccharides - QA-Tri(X / R)-F - QA-Trix-F or QA-TriR-F, QA glycosylated at C-28 and C-3 positions - QA-Tri(X / R)-FR - QA-Trix-FR or QA-TriR-FR, QA glycosylated at C-28 and C-3 positions - QA-Tri(X / R)-FRX - QA-Trix-FRX or QA-TriR-FRX, QA glycosylated at C-28 and C-3 positions - QA-Tri(X / R)-FRXA - QA-Trix-FRXA or QA-TriR-FRXA, QA glycosylated at C-28 and C-3 positions - QA-tri(X / R)-FRXX - QA-triX-FRXX, QA-triX-FRXX or QA-triR-FRXX, QA glycosylated at C-28 and C-3 positions - QA-tri(X / R)-FRX(X / A) - QA-triX-FRXX, QA-triX-FRXA, QA-triR-FRXX or QA-triR-FRXA, QA glycosylated at C-28 and C-3 positions - QA-F - QA monoglycosylated at C-28 position - QA-FR - QA diglycosylated at C-28 position - QA-FRX - QA triglycosylated at C-28 position - QA-FRXA - QA tetraglycosylated at C-28 position - QA-FRXX - QA tetraglycosylated at C-28 position - QA-FRX(X / A) - QA-FRXX or QA-FRXA, QA glycosylated at C-28 position - QA-triX-FRXA-Ac - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-kic acid acetylated at C-4 of D-fucose in C-28 chain - QA-triX-FRXA-R-Ac - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-kic acid acetylated at C-4 of D-fucose in C-28 chain together with the rhamnose moiety attached to C-3 of D-fucose in C-28 chain - QA-Trix-FRXA-G - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-Kyrinic acid, glycosylated at the C-3 position of the C-28 rhamnose moiety of the core C-28 rhamnose moiety with rhamnose. - QS-7 (or QA-Trix-FRXA-GR-Ac) - 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranuronate}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-Kyrinic acid, acetylated at the C-4 position of the D-fucose of the C-28 chain of the core C-28 chain, together with the rhamnose moiety attached to the C-3 position of the D-fucose of the C-28 chain and the glucose moiety attached to the C-3 position of the C-28 rhamnose moiety of the core C-28 chain. Qs_0283850 - Q. saponaria QA-diα-1,3-L-rhamnosyltransferase Qs_0283870 - Q. saponaria QA-diβ-1,3-D-xylosyltransferase Qs-28-O-ApiT4 - Kyrinic acid 28-O-fucoside[1,2]-rhamnoside[1,4]xyloside[1,3]apiosyltransferase Qs-28-O-FucT - Kyrinic acid 28-O-fucosyltransferase Qs-28-O-RhaT - Kyrinic acid 28-O-fucoside[1,2]-rhamnosyltransferase Qs-28-O-XylT3 - Kyrinic acid 28-O-fucoside[1,2]-rhamnoside[1,4]xylosyltransferase Qs-28-O-XylT4 - Kyrinic acid 28-O-fucoside[1,2]-rhamnoside[1,4]xyloside[1,3]xylosyltransferase Qs-3-O-GalT - Q. saponaria QA-mono-β-1,2-D-galactosyltransferase Qs-3-O-RhaT - Q. saponaria QA-di-α-1,3-L-rhamnosyltransferase Qs-3-O-RhaT / XylT - Q. saponaria QA-dual β-1,3-D-xylosyltransferase / α-1,3-L-rhamnosyltransferase Qs-3-O-XylT - Q. saponaria QA-di-β-1,3-D-xylosyltransferase QS-7-AcetylT - Kira acid 28-O-fucoside[1,4]acetyltransferase (also known as QsACT-19’). QS-7-GlcT - Kira acid 28-O-fucoside[1,2]-rhamnoside[1,3]glucosyltransferase (also known as QsUGT-BI) QS-7-RhaT - Kira acid 28-O-fucoside[1,3]rhamnosyltransferase (also known as QsUGT-23500) QsAXS1 - UDP-D-apiose / UDP-D-xylose synthase QsACT-19’ - Kira acid 28-O-fucoside[1,4]acetyltransferase (also known as Qs-7-AcetylT) QsbAS - Q. Saponaria β-amyrin synthase QsCSL1 - Q. saponaria cellulose synthase-like enzyme (Kira acid 3-O-glucuronosyltransferase) QsCslG2 - Q. saponaria cellulose synthase-like enzyme (Kira acid 3-O-glucuronosyltransferase) QsCYP716-C-28 - Q. saponaria Kira acid C-28 oxidase QsCYP716-C-16α - Q. saponaria Kira acid C-16α oxidase QsCYP714-C-23 - Q. saponaria Kira acid C-23 oxidase QsFSL-1 - An enzyme from Q. saponaria that increases the production of fucosylated saponins QsFSL-2 - An enzyme from Q. saponaria that increases the production of fucosylated saponins QsFucSyn - An enzyme from Q. saponaria that increases the production of fucosylated saponins QsUGT-BI -, kierraic acid 28-O-fucoside [1,2]-rhamnoside [1,3] glucosyltransferase (also known as QS-7-GlcT) QsUGT-0023500 - Kierraic acid 28-O-fucoside [1,3] rhamnosyltransferase (also known as QS-7-RhaT) Rhap - L-rhamnopyranose SoFSL-1 - An enzyme from S. officinalis that increases the production of fucosylated saponins UDP-sugar - Uridine diphosphate sugar UGT - UDP-dependent glycosyltransferase Xylp - D-xylopyranose

[0198] References Fleck JD, Betti AH, da Silva FP, Troian EA, Olivaro C, Ferreira F, Verza SG. 2019. Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities. Molecules 24(1). Kensil C R, Patel U, Lennick M, and Marciani D, 1991. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex, J Immunol. 146 (2) 431-437. Louveau T, Osbourn A. 2019. The Sweet Side of Plant-Specialized Metabolism. Cold Spring Harb Perspect Biol (In Press). Reed J, Osbourn A. 2018. Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant Cell Reports. Reed J, Stephenson MJ, Miettinen K, Brouwer B, Leveau A, Brett P, Goss RJM, Goossens A, O'Connell MA, Osbourn A. 2017. A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules. Metab Eng 42: 185-193. Sainsbury F, Thuenemann EC, Lomonossoff GP. 2009. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7(7): 682-693. Stephenson MJ, Reed J, Brouwer B, Osbourn A. 2018. Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale. Journal of visualized experiments : JoVE (138): 58169.

[0199] P202303GB - Attachment A

[0200] A nucleic acid sequence encoding an enzyme according to SEQ ID NO: 1 to SEQ ID NO: 2. *

[0201] SEQ ID NO: 2 - Fucosyltransferase enzyme (Qs-28-O-FucT) capable of transferring β-D-fucopyranose to the C-28 position of the chiral acid. MENGRVYKSHVVVLAFHGQGHIVPLIQLSRRLAWKGIKITFATTHSCTKAIQTGSDSISLLSIYDDITDGGFQGEGGFKGFLQRFEASTTRILHEFVKNHENSKNPVKCLIYDANLIWALEMAKQLGIATAAFVFPSWAAIATYYPFYLEVYADQQIKKVDPFTMPDLPPQLGLPNMASLGSDSGQHSPILKLMLQQLENFGKADWILSHAFEQFEQEVLDWMRNISPVTTIGPTLPSVYLDGRLKDDTDYGYNLYKPDSDTCMKWLDTKETESVVYISFGSVADLIPEQMTEITNSLKKMSSNFLWVVKETEKNNLPSSFVEETKEKGLVVTWCPQLKVLSHPAVGCFITHCGTNSIFESVCFAVPMVGMPQFCDQMPNAYFMEKVWKVGVRPSLDDNGVVTGEEIERCIKVVTEGESGQEIKKKLVQWKELAKEAVDEGGSSDKHIDEFIAGITT *

[0202] SEQ ID NO: 3 - Nucleic acid sequence encoding the enzyme according to SEQ ID NO: 4.

[0203] A rhamnosyl transferase enzyme (Qs-28-O-RhaT) with SEQ ID NO: 4 that can transfer α-1,2-rhamnopyranose to QA-F. MAKTDKQLHIAMFPWLAMGHIFPNFELAKLFAQKGHSITLISTPRNISRLPQIPTHLEQLIKLVSLPILPKHKANLPENAESTMDVTPNKVPYLKMAYDGLQESLTQLLKSSAPDWILYDFAADWLPPLVHSLQIRCVFFVVSPAWNLCFFDTPKPQLGSAAVFRTKPEDYLRPPSWVPFHSNIGLKLHEVKKMFEGVSDKETGVTVSFNFNKAVSSCDLFSFRSCYELESEWLNLVEDIYKRPVVPVGVIPPSFQVRIVNEEDNKPEWLKIQSWLDKQEQGSVVYIAFGSELKLGQQDLTELALGLELSGLPFFWALRKQQDSSSVDLPDGFEDRVSDRGVVCRDWVPQLKILAHGSIGGYLTHCGSGSVIEGLHFGRVLVMLPYLLDQALYARVLEEKKLGVEIPRNEQDGSFTRSSVAKSVKLAIVDEGGSIYRDKAKEMGLVFSDKDRHEQYIENFLQHLQHKREPFQI *

[0204] A nucleic acid sequence encoding an enzyme according to SEQ ID NO: 5 - SEQ ID NO: 6.

[0205] Array number 6 - A xylosyltransferase enzyme (Qs-28-O-XylT3) capable of transferring β-1,4-D-xylopyranose to QA-FR MAAAAPNHRLHIAFFPWLAFGHINPFFELAKLIAQKGHHISFISTPRNIQRLSQVPPQLADSIDLVSLPVIHNSNLPENAESTMDIPPDKTPYLGMLHDSLKEPLTQFLQTHSPDWILYDFSAGWLAAIVEDLGISHGYFSIIPCWNIGFNGRQMNGFQKPDISLPSAVSLKKYEVKKIMDLVKSFPKILDESATKSIASHSTCEVIFIRNCPEIEADWFDYTSKIFDKPVVPVGVVPPSVHITNKEKDEHFNKWLEIKEWLDQQDRGSVIYIAFGTESLPNQDEITMLAQGLELCGLPFFWALRKSNVASDQPNSDSVELPEGFEEQTKGRGIVWTSWAPQQRILGHNSIGGFVTHCGWSSVIEGIHYGRPLIMFPLTVEQSLNARILGEKKLGMEVPREDDGSFTGEVVAETLKLVLLDQDGKVYRDKVTEMSKVFGDKDKHEKYMGDLLEFFKNYRSLKRN *

[0206] Array number 7 - A nucleic acid sequence encoding an enzyme according to SEQ ID NO: 8

[0207] SEQ ID NO:8 - A xylosyltransferase enzyme (Qs-28-O-XylT4) capable of transferring β-1,3-D-xylopyranose to QA-FRX MDSTHLQPATTPLKIHFIPFISPGHIIPLSELARIFASRGEHVTIITTPFNADLLQKSIDEDRDSGEHIGIHTVEWSATELGLPHGIENLSNVTDLETAIKLHRALMLMQKQMEDFMTRNPPDCIIADTFYPWASEFANRMGIPRLIFYPWSTFALCLMESIRSPDSPHRRLSSDSDPFVVPGLPHPIILTRSQLPEHDRKDIADPAAQLMDQHKETEMKSYGIILNNFAEIETEYTEHYKKITGHKVWHIGPAAAIVHRNAKEKAERVFKSDEHDNNLVINWLNSKEPNSVVYVCFGSGCQFPDKQLYEIACGLELSGHQFVWVVRGKDKQIDVNDDGEKTWLPKGFEERMKTENKGLIVRGWAPQVLVLDHPSLGCFLTHCGWNSTIEGITAGVPLITWPVFAEQFYNEKLITQVHGNGVVVGSEEWIMLFTVAKSLVSRDKIENAVRKIMDGGDEAVQIRRRARELGEKAWKAASTGGSSYNNLTAAIEDLKRLREDRSKLKTKTI *

[0208] SEQ ID NO:9 - A nucleic acid sequence encoding an enzyme according to SEQ ID NO:10.

[0209] Array No. 10 - Apiosyltransferase enzyme (Qs-28-O-ApiT4) capable of transferring β-1,3-D-apiofuranose to QA-FRX. MDSTHLQPATTPLKIHVIPFIAPGHIIPLSELARIFASRGEHVTIITTPFNADLLQKSIDEDRDSGEHIGIHTVEWSATELGLPHGVENLSNVTDLETGIKLHRALVLMQKQMEDFMTRNPPDCIIADTFYPWASEFANRMGIPRLIFFPGCTFALCLMESIRSPDSPHRRLSSDSDPFVVPGLPHPIILTRSQLPEHDREDIADPAAQFMDQCKEAAMKSYGIILNNFAEIETEYTEHYKKITGHKVWHIGPAAAIVHRNAKEKAEGLFKSDEHDNNLVINWLNSKEPNSVVYVCFGSGCQFPDKQLYEIACGLELSGHQFIWVVRGKDKQIDVNDDEEKTWLPKGFEERMKTENKGLIVRGWAPQVLVLDHPSLGCFLTHCGWNSTIEGITAGVPLITWPVYSEQFYNEKLITQVHGNGVGVGSEEWIMLFSVAKSLVSRDKIENAVRKIMDGGDEALEIRRRARELGEKARKAASIGGSSDNNLTAAIEDLKRLREDRSKLKTKTI

[0210] Array No. 11 - Nucleic acid sequence encoding an enzyme according to Array No. 12. ATGGCAGAAGCAACGCAGAGGTATGCTGTTGTGACAGGATCTAATAAGGGAATTGGATTTGGGATATGCAAGCAGTTGGCTTCTAAGGGAATCAAAGTAGTGTTAACAGCTAGAGATGAGAAGAGAGGTCTTGAAGCAGTTGAGAAATTGAAAGAAATTAGTCTGGCTGGTCATGTGGTTTTTCATCAACTCGATGTGTCTGATCCTGCTAGTGTTACTAGCCTTGAAGATTTCATCAAAACCCAGTTTGGGAAGCTAGATATTCTGGTAAACAATGCTGGGATAACAGGAACAACTGTAGATGCTGATGCTTTAGCAGCTTCAGGCTTCGGTACAGGGGGTGAACGTAAGCCTATTGATTGGAGTAAGTTAGTGATACAGACTTATGAATCAGTTGAAAAAGCTTTCAACACAAACTATTACGGTGGCAAAAGAATGACAGAAGCACTTATACCCCTCCTCCAGCTATCAGACTCACCCAGGATTGTTAATGTTTCCTCTGCTATGGGACAGTTAGAGAATATACCTAGTGGATGGGCAAAGGAAGTGCTCACAGATGTTGATAACCTAACAGAAGGAAAATTGGATGAGGTTTCAACCCAGTTTTTGAAAGATTTCAAAGAGGGTTCATTGGAAACCAAAGGCTGGCCTAGTCTTATGTCTTCTTATATAGTCTCAAAAGCTGTTTTAAATGCCTACACAAGGATTCTTGCTAAGAAATACCCAGCTTTCTGCATCAATTGTGTAGATCCTGGCTATGTGAAGACAGACATAAACCATCATACTGGCCAATTAAGTGTTGATGAAGGTGCTGAAAGTCCTGTAAGACTGGCCTTGCTGCCTAATGGTGGTCCTTCTGGCGTGTTCTTCTCCAGGACAGAAGAAGCACCATTTTGA

[0211] SEQ ID NO: 12 - Oxidoreductase enzyme (QsFucSyn) capable of enhancing the activity of fucosyltransferase MAEATQRYAVVTGSNKGIGFGICKQLASKGIKVVLTARDEKRGLEAVEKLKEISLAGHVVFHQLDVSDPASVTSLEDFIKTQFGKLDILVNNAGITGTTVDADALAASGFGTGGERKPIDWSKLVIQTYESVEKAFNTNYYGGKRMTEALIPLLQLSDSPRIVNVSSAMGQLENIPSGWAKEVLTDVDNLTEGKLDEVSTQFLKDFKEGSLETKGWPSLMSSYIVSKAVLNAYTRILAKKYPAFCINCVDPGYVKTDINHHTGQLSVDEGAESPVRLALLPNGGPSGVFFSRTEEAPF *

[0212] A nucleic acid sequence encoding an enzyme according to SEQ ID NO: 13 - SEQ ID NO: 14.

[0213] SEQ ID NO: 14 - Enzyme (QsAXS1) capable of enhancing the activity of apiosyl transferase. MPTLYKKAGLMASASRVDLDGNQIKPMTICMIGAGGFIGSHLCEKIMAETPHKVLALDVYNDKIKHLLEPDSLQWKDRIQFHRINIKHDSRLEGLIKMADLTINLAAICTPADYNTRPLDTIYSNFIDALPVVKYCSENNKRLIHFSTCEVYGKTIGSFLPKDSPLLKDPEYFVLKEDASPCIFGPIEKQRWSYACAKQLIERLVYAEGAENGLEFTIVRPFNWIGPRMDFIPGIDGPSEGVPRVLACFSNNLLRGEPLKLVDGGQSQRTFVYIKDAIEAVLLMIENPARANGHIFNVGNPHNEVTVRQLAEMMTEVYSKVSGEPSLEVPTIDVSSKEFYGEGYDDSDKRIPDMTIINRQLGWNPKTSLWDLLESTLTYQHRTYAEAIKKSIAKPVAS *

[0214] SEQ ID NO: 15 - Nucleic acid sequence encoding an enzyme according to SEQ ID NO: 16.

[0215] Accession No. 16 - Translated nucleotide sequence of AstHMGR (Avena strigosa truncated HMG-CoA reductase) (424 aa): MAPEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDGFDYDSILGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCLIASTNRGCKAIAESGGASSVVYRDGMTRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFTCSTGDAMGMNMVSKGVQNVLDYLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSVVCEAVIREEVVHKVLKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVESSQCITMLEAVNDGRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVLAGELSLISAQAAGHLVQSHMKYNRSSKDMSKIAC

[0216] Nucleic acid sequence encoding an enzyme according to Accession No. 17 - Accession No. 18

[0217] Array number 18 - Enzyme (QsbAS) involved in the synthesis of β-amyrin from 2,3-oxide squalene MWRLKIAEGGSDPYLFSTNNFVGRQTWEFEPEAGTPEERAEVEAARQNFYNNRYQVKPCDDLLWRYQFLREKNFKQTIPPVKVEDGQEITYEMATTSMQRAARHLSALQASDGHWPAQIAGPLFFMPPLVFCVYITGHLNTVFPSEHRKEILRYMYYHQNEDGGWGLHIEGHSTMFCTALNYICMRILGEGPEGGQDNACARARMWILDHGGVTHIPSWGKTWLSILGLFEWSGSNPMPPEFWILPSFLPMHPAKMWCYCRMVYMPMSYLYGKRFVGPITPLIVQLREEIHTQNYHEINWKSVRHLCAKEDIYYPHPLIQDLIWDSLYILTEPLLTRWPLNKLVRERALQVTMKHIHYEDENSRYITIGCVEKVLCMLACWVDDPNGDAFKKHLARVPDYVWVSEDGITMQSFGSQEWDAGFAVQALLASNLTEELGPALAKGHDFIKQSQVKDNPSGDFKSMYRHISRGSWTFSDQDHGWQVSDCTAEGLKCCLLLSMLPPEIVGEKMEPQRLFDSVNVLLSLQSKKGGLAAWEPAGAQDWLELLNPTEFFADIVVEHEYVECTGSAIQALVLFKKLYPGHRKKEIDSFITNAVRFLENTQTADGSWYGNWGVCFTYGCWFALGGLAAAGKTYNNCPAIRKAVNFLLTTQREDGGWGESYLSSPKKIYVPLEGSRSNVVHTAWAMMGLIHAGQAERDSTPLHRAAKLIINYQLENGDWPQQEITGVFMKNCMLHYPMYRNIYPMWALAEYRRRVPLP *

[0218] Nucleic acid sequence encoding an enzyme according to Array number 19 - Array number 20

[0219] Enzyme (QsCYP716-C-28) involved in the synthesis of oleanolic acid from sequence number 20 - β - amyrin MEHLYLSLVLLFVSSISLSLFFLFYKHKSMFTGANLPPGKIGYPLIGESLEFLSTGWKGHPEKFIFDRMSKYSSQIFKTSILGEPTAVFPGAVCNKFLFSNENKLVNAWWPASVDKIFPSSLQTSSKEEAKKMRKLLPQFLKPEALHRYIGIMDSIAQRHFADSWENKNQVIVFPLAKRYTFWLACRLFISVEDPTHVSRFADPFQLLAAGIISIPIDLPGTPFRKAINASQFIRKELLAIIRQRKIDLGEGKASPTQDILSHMLLTCDENGQYMNELDIADKILGLLVGGHDTASAACTFIVKFLAELPHIYEQVYKEQMEIAKSKVPGELLNWEDIQKMKYSWNVACEVMRLAPPLQGAFREAITDFVFNGFSIPKGWKLYWSANSTHKSPDYFPEPDKFDPTRFEGNGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKLVPDEKIVVDPMPIPAKGLPVRLYPHKA *

[0220] Nucleic acid sequence encoding an enzyme according to sequence number 21 - sequence number 22.

[0221] Accession No. 22 - Enzyme (QsCYP716-C-16α) involved in the synthesis of echinocystic acid from oleanolic acid MIYNNDSNDNELVISSVQQPSMDPFFIFGLLLLALFLSVSFLLYLSRRAYASLPNPPPGKLGFPVVGESLEFLSTRRKGVPEKFVFDRMAKYCRDVFKTSILGATTAVMCGTAGNKFLFSNEKKHVTGWWPKSVELIFPTSLEKSSNEESIMMKQFLPNFLKPEPLQKYIPVMDIITQRHFNTSWEGRNVVKVFPTAAEFTTLLACRVFLSVEDPIEVAKISEPFEILAAGFLSIPINLPGTKLNKAVKAADQIRDAIVQILKRRRVEIAENKANGMQDIASMLLTTPTNAGFYMTEAHISEKILGMIVGGRDTASTVITFIIKYLAENPEIYNKVYEEQMEVVKSKKPGELLNWEDVQKMKYSWCVACEAMRLAPPVQGGFKVAINDFVYSGFNIRKGWKLYWSAIATHMNPEYFPEPEKFNPSRFEGKGPVPYSFVPFGGGPRMCPGKEYSRLETLVFMHHLVTRYNWEKVYPTEKITVDPMPFPVNGLPIRLIPHKHQ *

[0222] Accession No. 23 - Nucleic acid sequence encoding the enzyme according to Accession No. 24

[0223] Accession number 24 - Enzyme (QsCYP714-C-23) involved in the synthesis of kiralic acid from echinocystic acid. MWFTVGLVLVFALFIRLYSSLWLKPRATRIKLSNQGIKGPKPAFLLGNVAEMRRFQSKLPKSELKQGQVSHDWASKSLFPFFSLWSQKYGNTFVFSLGNIQVLYVSDHELVKEINQNTSLDLGKPKYLQKERGPLLGQGILTSNGQLWAYQRKIMTPELYKEKIKGMCELMVESVAWLVEEWGTKIQAEGGAADIRIDEDLRSFSGDVISKACFGSCYAGGREIFLRLRALQHQIASKALLMGFPGLKYLPIKSNREIWRLEKEIFQLIMKLAEDRKKEQHERDLLQIIIEGAKSSDLSSEAMAKFIVDNCKNVYLAGHETTAMSAGWTLLLLANHPEWQARVRDEILQVTEGRNPDFDMLHKMKLLTMVIQEALRLYPTVIFMSREALEDINVGNIQVPKGVNIWIPVVNLQRDTTVWGADANEFNPERFANGVNNSCKVPQLYLPFGAGPRICPGINLAMTEIKILLCILLTKFSFSVSPNYRHSPVFKLVLEPENGINVIMKKL *

[0224] Accession number 25 - Nucleic acid sequence encoding an enzyme according to Accession number 26.

[0225] SEQ ID NO: 26 - Enzyme involved in the synthesis of QA mono from D - quinic acid (QsCSL1). MKSPSNPNQKPILHTCTIQQPRATLNKIHSLIHFSAILVLFYYRITRLFFTDDFKVPKLLWTLMTISEFILAFIWVLIQPFRWRPVSRSVIPENMPKDISLPAVDVFVCTADPQKEPTVEVMNTILSAMALDYPAEKLAVYLSDDGGSAVTLYAIKEACCFAKMWLPFCNKYGIKSRCPEAYFSKLAADEWLHRSVEFVAEEKEVKANYEEFKRNVQKFGEQQENSRVVHDRHPHVEIIHNNWNNEDQAHEMPLLVYVSRERRPSHHPRFKAGALNTLLRVSGIISNSPYILVLDCDMYCNDPTSARQAMCFHLDPQLSKNLAFVQFPQIFYNASKNDVYDAQVRAAYQTKWQGMDGLQGPIFSGTGFYLKRKAMYGNPDQDDNCLLKPYKKFGMSGEFVESLKVLNEQDGTQKKLLDGFLQEAKLLASCAYETKTSWGKEIGFSYDCLIESTFTGYLLHCRGWISVYLYPKRPCFLGCCPTDMKDAMVQYTKWMSELFSIAISRFNPLLYGVARMSILQSLCYGSFTLAPILSFPLFLYGTVPQLCLLKGISLFPKVSDPWFAVFAAIFVSSLCQHWFEVLSCDGTFTTWCNEQRSWLIKSVSGSLFGVVGAILQRLGLKTKFSLSNKAMDKEKLEKYEKGKFNFQGAAMFMVPVSILVILNTFCFLGGFWKVIIMKNILDMFGQLSLSAYVLVLSCPVLEGMLTRISKKMV *

[0226] SEQ ID NO: 27 - Nucleic acid sequence encoding the enzyme according to SEQ ID NO: 28.

[0227] Accession number 28 - Enzyme (QsCslG2) involved in the synthesis of QA mono from Kira acid. MATVSSLHTCTVQQPRAAINRIHIFLHFIAILFLFYYRVTGLFYDNAVPTLAWSLMTLAELIFAFVWVLSQAFRWRPVLRSVIPERIPKDVRLPAVDILICTADPLKEPTVEVMNTVLSAMALDYPAENLAVYLSDDGGSPVTLFAMKQVGPFAKLWLPFCNKYGIKTRHPESFFSAFADDERLHRSDEFRAEEEAIKDKYEEFKRTIEKYGGEGKNSHVVQDRPPHVEIIHDTRKIRENSEDQAVPLLVYVSREKRPSYNSRFKAGALNTLLRVSGVISNSPYVLVLDCDMYCNDPTSARQAMCFHLDPQMSRTLSFVQFPQVFYNVSKNDIYDGQARAAFKTKWQGMDGLRGPLLSGTGFYLKRKSLYGSPNQEDDCLLEPHKNFGKCDKLIESVKVIYERDVSIKADSSDAILQDAKQLASCPYETNTSWGKEVGFSYDCLLESTFTGYLLHCRGWTSVYLYPKKPCFLGCTPVDMKEAMVQYTKWISELFLLAISRFNPLTFGISRMSILQSMCYGYLTIMPILSVAMIFYATVPQLCLLRGVPLFPKVSDPWFAVFLAIFVSSLCQHLIEVLTSDGTLKTWWNEQRNWVIKSGSGSVFGALSGILKWFGMKIKFGLSNKAVDKEKLEKYEKGKFDFQGAAMFMVPLTISVILNTLCLIGGLWRVITLKNFEEMSGQFIISLYFLALSYPILEGLLRKGKGKA

[0228] Accession number 29 - Nucleic acid sequence encoding an enzyme according to Accession number 30.

[0229] SEQ ID NO: 30 - Enzyme involved in the synthesis of QA-J from QA-Mono (Qs-3-O-GalT). MVESPADHDVLKIIVLPWVTSGHMIPMVDAARLFAMHGADVTIITTPANALTFQKSVDRDFNSGRLIRTHTLKFPAAEVGVPEGVENFNNTSPEMTSKVYLGVSMLREPTQQLIEDLRPDCLITDMFYPWAVDVADKLGIPRLIFQGPGSFGLSAMHSIKQYEPFKSVTSDTETFPLPGLPHKVEMTRLQIPKWVREPNGYTQLMGRVKDSERRSYGSLVNSFYDFEGPYEEHYRKATGQRVWSIGPVSVWVNQDAADKVGRGQDLVAEDQNSWLNWLNSKEKNSVLYVSFGSMAKFPSAQLLEIAHGLEASGHSFIWVVRKVDGDDDVDVWLPDFEKKMKENNKGFIIRNWAPQLLILDHPAIGGLLNHSGWNSVLEGATAGLPMITWPLYAEHFYNERLVLDVLKIGVPVGVKEWKNLHEVGELVRRDAIAKAIKLLMGSGEEAEVMRKKAKELGVGAKKGIQVGGSSHTNLIAVIDELKSLKKSRIQGV *

[0230] SEQ ID NO: 31 - Nucleic acid sequence encoding an enzyme according to SEQ ID NO: 32.

[0231] Enzyme (Qs-3-O-RhaT / XylT) involved in the synthesis of QA-Tri R or QA-T Tri X from array number 32 - QA-Ji. MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASGYHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIVADMFTSWTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPDGTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLIHRNDEDKIQRSHKTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDEDNDDVGNKWLPAFEERIKKENKGMILRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKAAQDHES *

[0232] Nucleic acid sequence encoding an enzyme according to array number 33 - array number 34.

[0233] Enzyme (Qs_0283850) involved in the synthesis of QA-tri R from array number 34 - QA-ji. MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKGKTSGYHVNIHAVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPDCIVADMFNRWTSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDGAFSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGPLQLIYQNDDPKVQRSQKTAVVSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVGNKWLPDFEERIKRENKGIIFRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDCGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKVA *

[0234] Nucleic acid sequence encoding an enzyme according to array number 35 - array number 36.

[0235] Array number 36 - Enzyme (DN20529_c0_g2_i8) involved in the synthesis from QA-J to QA-Tri R. MVSGDDTVSRPLIVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKRKNSGYHVNIHTVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPDCIVADMFNRWTSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDGAFSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGPLQLIYQNDDPKVQRSQKTAVLSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVGNKWLPGFEERIKRENKGIIFRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKAAMKS *

[0236] Array number 37 - Nucleic acid sequence encoding the enzyme according to Array number 38.

[0237] Enzyme (Qs_0283870) involved in the synthesis of QA-Tri X from Array No. 38 - QA-Ji. MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASGYHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIVADMFTSWTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPDGTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLIHRNDEDKIQRSHKTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDEDNDDVGNKWLPAFEERIKKENKGMILRGWAPQMLILNHPAIGGFMTHCGWNAAVEALSSGVPIITFPVFSDQFYNERLISQVHKCGVGVGTEAWSYAFDAGKNPVGREKIMTAVKKILDGGEEAEGMRKRARELKEIAKRSVEEGGSSYNNLTAMIQDLKEFRANNGKAAQDHES *

[0238] Array No. 39 - Acanthocystis turfacea chlorella virus 1 UDP-D-glucose 4,6-dehydratase (ATCV-1) coding sequence (1053bp): Note: This sequence has been codon-optimized for expression in N. benthamiana. The original sequence can be referenced as Genbank ID: NC_008724.1 (see locus tag ATCV_z554R).

[0239] Accession number 40 - Translated nucleotide sequence (350 aa) of UDP-D-glucose 4,6-dehydratase (ATCV-1) of Acanthocystis turfacea chlorella virus 1: MNSQEYTPKSVLVTGGAGFIGSHVVMKLVQRYPECKVVVLDKMDYCATLNNLATVRDAPNFKFVKGDIQSTDLLAHVLKQEKIDTIMHFAAQTHVDNSFGNSLAFTMNNVYGTHVLLECARLYGGVQRFINVSTDEVYGESSLGKKEGLDEHSSLEPTNPYAAAKAGAEMMARAYHTSYKLPVIVTRGNNVYGPHQFPEKMIPKFILRATRGLDLPIHGDGGALRSYLYVDDVAEAYITILLKGNVGETYNIGTQKERSVVDVAHDICKIFNRDSDTAIWHVKDRAFNDRRYFISDKKLLDLGWQEKTTWEDGLKQTVGWYLQHATRSYWDHGNMELALDAHPTLQVPKF *

[0240] Accession number 41 - Coding sequence (693 bp) of NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AaFCD) of Aggregatibacter actinomycetemcomitans: Note: This sequence was codon-optimized for expression in N. benthamiana. The original sequence can be referenced as Genbank ID: AB002668.1 (sequence 15271..15963 bp). ATGATAATTGGCAACGGTATGCTGGCAAAAGCCTTTGAATCATTCCATAAGCGAACTTACAATTACATAATATTTGCATCCGGAGTGAGCAACTCAAACGAAACTTCCTTCGAGAATTTCAACAGAGAGAAGGAATTGCTTCTTGAAGTCCTGGAGCAATATAAAGACAAAACTATCGTTTACTTTAGTTCCTGCTCCATATACGATTCTAGTTTGACGAATTCTTTGTATGTCTACCACAAAATGTGTATGGAGAGACTGGTGCGTGAAAACTCCAAGAATTATCTCATAGCCCGTCTCCCCCAAGTTATTGGTAAAACGTATTCACCAACCATTGTCAACTTCCTTTTTAACAAAATCAAAAATAGGGAGTGTTTCAGCATATTCGGAAAAGCTCACCGAAATTTTATCGACGTGGATGATGTCGTTAAGGTCACCAATTACTTATTGAAGGAGGGTCTGTTCATTAACAGTATTGTGAACTTGGCAAGCACGCACCATACCTCCATGTACGAATTAATCTTATATTTGGAAAAAATAAGTAATCAACGTGCCTTCTATAATGTTGAGAACAAAGGGTCTAGGTACTTTATTGATGTTTCAATACTGCAGGATGTTTATCAGAAGCTGGGGATCAAATTTGACAAAGATTACGTAGAAAAGGTTATCAACAAGTACTACGCTATTAAGTAA

[0241] Sequence number 42 - Translated nucleotide sequence (230aa) of Aggregatibacter actinomycetemcomitans NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AaFCD): MIIGNGMLAKAFESFHKRTYNYIIFASGVSNSNETSFENFNREKELLLEVLEQYKDKTIVYFSSCSIYDSSLTNSLYVYHKMCMERLVRENSKNYLIARLPQVIGKTYSPTIVNFLFNKIKNRECFSIFGKAHRNFIDVDDVVKVTNYLLKEGLFINSIVNLASTHHTSMYELILYLEKISNQRAFYNVENKGSRYFIDVSILQDVYQKLGIKFDKDYVEKVINKYYAIK *

[0242] Accession number 43 - Anoxybacillus tepidamans NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AtFCD) coding sequence (927 bp): Note: This sequence was codon-optimized for expression in N. benthamiana. The original sequence can be referenced as Genbank ID: AY883421.5 (sequence 13209-14135 bp). ATGAAGAGGATACTGATACTCGGATGCGGTTACCTGGGTTTAAATCTCGCAAACTATTTTTGTAAAAAAAATTATGATGTCTCAGTGATAGGGAGAAAGTCTGTCTATAGCAATTTTTTGGAAGAGGAGATAGAGTTCATAGAAGATGATATCAAAAATATAAATAGTTATAAGCACATGTTTAATGAGGAGACAACCGTCATTTACGCCATAGGAAGTATTAACGCAAATAACTATTTTATGGACCTGAGGAATGATATAGAAAACTCATACATCCCCTTCATTAACCTCCTTAACTTTCTTTCCGAAAAGTATATTCAAAAGTTCGTCTTTCTCTCTTCAGCCGGAACAGTCTATGGGAACGTGAATAAGAATTATATAAGCGAGAATGAGATTCTTAACCCAATTTCAATCTATGGTTTGCAGAAAGCCTTCTTTGAACAACTGATAAGGATTAAAAACAATGAGGCTAGCCATTTCAGGTATTTGATCTTCAGAATATCTAACCCCTATGGGGGAATCAACATTCCGAACAAGAATCAGGGAATTATTCCGACGTTAGTGTACAAAGCCGTGAACAATGAGCCTTTCGAACTTTGGGCATCAATCAATACCATCCGTGATTATATTTACATCGATGACCTTAGCGAATTGATCTACAAAACAATCTATCTGGACATTTATAACGAGACCCTCAATCTCGGGTCCGGTAAAGGAACATCAATCAAGCAACTCATTAGCCTCGTGGAGGAGATTTTGGGAAAGAAGATCACTATTCTTGAAAAGCCCCCCATAAAGACTAACGTTTTGAAAAATATACTTGATATTTCTAAGCTCGTCAACACCGTAGGCTACGAACCAAAGATCAGCATTGAAGAGGGTATTAGCCGTTACATCAACACTATTTTAACGAAGAACATTTTTTAA

[0243] Sequence number 44 - Translated nucleotide sequence (308aa) of Anoxybacillus tepidamans NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AtFCD): MKRILILGCGYLGLNLANYFCKKNYDVSVIGRKSVYSNFLEEEIEFIEDDIKNINSYKHMFNEETTVIYAIGSINANNYFMDLRNDIENSYIPFINLLNFLSEKYIQKFVFLSSAGTVYGNVNKNYISENEILNPISIYGLQKAFFEQLIRIKNNEASHFRYLIFRISNPYGGINIPNKNQGIIPTLVYKAVNNEPFELWASINTIRDYIYIDDLSELIYKTIYLDIYNETLNLGSGKGTSIKQLISLVEEILGKKITILEKPPIKTNVLKNILDISKLVNTVGYEPKISIEEGISRYINTILTKNIF *

[0244] Sequence number 45 - Coding sequence (951bp) of Escherichia coli NDP-4-keto-6-deoxyglucose 4-ketoreductase (EcFCD): Note: This sequence was codon-optimized for expression in N. benthamiana. The original sequence can be referenced as Genbank ID: AY528413.1 (sequence 3156-4106bp). ATGGATGCTCGTAAAAATGGGGTATTAATAACCGGTGGAGCTGGGTTCATAGGTAAAGCCTTAATAACTGAAATGGTCGAACGTCAAATTCCCCTGGTGTCATTTGACATCAGCGATAAGCCCGACAGTTTGCCAGAGCTTTCCGAATATTTCAACTGGTATAAATTCTCATACCTTGAGAGTTCACAGAGGATTAAAGAGCTTCACGAAATAGTTTCCAGGCATAACATCAAAACGGTCATCCATTTAGCTACAACTATGTTTCCCCACGAATCCAAAAAGAACATCGATAAGGATTGCTTAGAAAACGTTTATGCCAACGTGTGTTTCTTTAAGAATTTATATGAAAACGGCTGTGAAAAAATTATCTTCGCCTCATCAGGTGGCACCGTATATGGGAAGTCTGATACACCCTTCTCCGAAGACGATGCCCTGCTTCCCGAAATTTCCTACGGACTGTCCAAGGTTATGACTGAAACTTATCTCCGATTCATAGCCAAGGAATTGAATGGGAAGTCCATCTCTCTCAGAATATCTAACCCCTATGGTGAGGGGCAAAGGATTGACGGGAAACAAGGAGTCATTCCAATTTTCCTCAATAAAATCAGCAACGACATCCCCATCGACATCATTGGCTCTATCGAATCAAAGCGAGACTACATTTATATTTCAGATCTCGTACAAGCTTTCATGTGCTCTCTGGAATATGAAGGTCACGAAGACATATTTAATATAGGTTCTGGGGAAAGCATAACTCTGAAGAAATTGATCGAGACGATTGAGTTCAAGCTGAACAAGAAGGCTGTGATTGGATTTCAAGATCCGATCCACACCAATGCCAATGGTATAATTCTCGACATCAAACGAGCCATGGCAGAACTCGGCTGGAGGCCCACCGTGGTCCTGGATGATGGCATCGATAAATTAATCAAGAGCATTCGATGCAAGTAA

[0245] Sequence number 46 - Translated nucleotide sequence (316 aa) of Escherichia coli NDP-4-keto-6-deoxyglucose 4-ketoreductase (EcFCD): MDARKNGVLITGGAGFIGKALITEMVERQIPLVSFDISDKPDSLPELSEYFNWYKFSYLESSQRIKELHEIVSRHNIKTVIHLATTMFPHESKKNIDKDCLENVYANVCFFKNLYENGCEKIIFASSGGTVYGKSDTPFSEDDALLPEISYGLSKVMTETYLRFIAKELNGKSISLRISNPYGEGQRIDGKQGVIPIFLNKISNDIPIDIIGSIESKRDYIYISDLVQAFMCSLEYEGHEDIFNIGSGESITLKKLIETIEFKLNKKAVIGFQDPIHTNANGIILDIKRAMAELGWRPTVVLDDGIDKLIKSIRCK *

[0246] Sequence number 47 - Nucleic acid sequence encoding the QsFSL-1 enzyme according to sequence number 48. QsFSL-1 ATGGCAGAAGCAACAGAGAGGTATGCTGTTGTGACAGGATCTAATAAAGGAATTGGATTTGGGATATGCAAGCAGCTGGCTTCTAAGGGGATTACAGTAGTGCTAACAGCTAGAGATGATAAGAGAGGTCTTGAAGCAGTTGAGAAATTGAAAGAATTTGATCTGCATGGTCATGTGCTTTTTCATCAACTTGATGTGTCTGATACAGCTAGTGTTACTAGCCTTGCAGATTTTATCAAAACCCAGTTTGGGAAACTAGATATCTTGGTAAACAATGCAGGTATAACTGGAACCACTGTAGATGCTGATGCTTTAGCATCTTCAGGCTATGGTACTGGGGGTGAACGTAAACCTATTGATTGGAATAAAATAGTGATAGAGACTTATGAATCAGTTGAAAAAGCTATCAACACCAACTATTATGGAGCCAAAAGAATGGCTGAAGCACTTATACCCCTTCTTCAAGTATCAGACTCACCAAGGATTGTTAATGCTTCCTCTCCTATGGCAAAGCTAGAGAATATTCCAAGTGGATGGGGTAAGGAAGTGCTAAGTGATGTTGATAGCCTAACAGAAGAGAAACTTGATGAGATGTTGACCCAATTATTGAAAGATTTCAAAGAGGGTTCATTAGAAACCAAAGGCTGGCCTACTCTTATGTCTTCGTATATAATCTCAAAAGCTGCTTTAAATGCCTACACAAGGATTCTTGCTAAGAAGTACCCATCTTTCTGCATCAATTGTGTAGACCCTGGTCATGTGAAGACTGACATAAATCGTCACACCGGCCACTTAAGTATTGATGAAGGTGCTGAAAGCCATGTGAGATTGGCCCTGCTGCCTGATGGTGGCCCTTCTGGACATTTCTTCTCCAGGACTGAAGAGACACCATTTTGA

[0247] SEQ ID NO: 48 - Oxidoreductase enzyme (QsFSL) capable of enhancing the activity of fucosyltransferase MAEATERYAVVTGSNKGIGFGICKQLASKGITVVLTARDDKRGLEAVEKLKEFDLHGHVLFHQLDVSDTASVTSLADFIKTQFGKLDILVNNAGITGTTVDADALASSGYGTGGERKPIDWNKIVIETYESVEKAINTNYYGAKRMAEALIPLLQVSDSPRIVNASSPMAKLENIPSGWGKEVLSDVDSLTEEKLDEMLTQLLKDFKEGSLETKGWPTLMSSYIISKAALNAYTRILAKKYPSFCINCVDPGHVKTDINRHTGHLSIDEGAESHVRLALLPDGGPSGHFFSRTEETPF *

[0248] A nucleic acid sequence encoding an enzyme according to SEQ ID NO: 49 - SEQ ID NO: 50. QsFSL-2 ATGGGTTCAGATGGAAGGGATGTAGCAGAGAGGTATGCAGTGGTTACAGGTGCAAACAAAGGCATAGGCCTAGAAACCGTGCGGCAACTAGCGTCTCACGGCATTACAGTTGTGTTGACAGCTCGAGATGAGAAGAGAGGGACTGAAGCCACAAGAAAGCTCCACCAGCTGGGTTTGTCAAATTTGATTTTCCATCAGCTGGATGTTTTAGACCCTGTTAGCATTCAGTCACTGGCCAAGTTCATCCAAGACAAATTTGGCAGGCTTGATATCCTGGTTAATAATGCTGGAGCATCTGGACTTGCAGCTGATGAGAAAGCTCTGAAGGCATTAAACATAGATAATGCAGCTTGGCTCTCAGGCAAGGCCGCCAATTTAGTTCAAGGAATTGTCACACATACCTATGAGCAAGGCGAAGAATGCATAAATACAAACTATTATGGTGTCAAAAGGGTGACGGAAGCTCTCCTACCGCTGTTACAACTTTCCCCTATAGGAGCAAGGATAATAAATGTTTCCTCTTGCAGGGGTGAGCTAAAGAGGATTCCAATGAACGTAAGAAATGAACTGGGCGACATCAAAGTTCTGACTGAAGGCAGAATAGATGCAATTTTGATGAAATTTCTACACGATTTTAAGGATAATGCACTTGAGTCCAACGGATGGACATTGATGGGGCCTGCTTATAGCATTTCGAAGGCCAGTCTCAATGCCTACACTAGACTTCTTGCCAAAAAGTACCCCGAGATGCTCATTAACTGTGTTCATCCTGGTTATGTCAACACAGATATGACTTGGCATAGAGGGATACTGACGGTAGAAGAGGGTGCTAAAGGCCCAGCCATGCTAGCTCTTTTGCAAGATGGAGGACCTACAGGTTGCTATTTTGATAGTACTCAACAGGCAGAATTTTAA

[0249] Accession No. 50 - Oxidoreductase enzyme (QsFSL-2) capable of enhancing the activity of fucosyltransferase MGSDGRDVAERYAVVTGANKGIGLETVRQLASHGITVVLTARDEKRGTEATRKLHQLGLSNLIFHQLDVLDPVSIQSLAKFIQDKFGRLDILVNNAGASGLAADEKALKALNIDNAAWLSGKAANLVQGIVTHTYEQGEECINTNYYGVKRVTEALLPLLQLSPIGARIINVSSCRGELKRIPMNVRNELGDIKVLTEGRIDAILMKFLHDFKDNALESNGWTLMGPAYSISKASLNAYTRLLAKKYPEMLINCVHPGYVNTDMTWHRGILTVEEGAKGPAMLALLQDGGPTGCYFDSTQQAEF *

[0250] Accession No. 51 - Nucleic acid sequence encoding an enzyme according to Accession No. 52. SoFSL-1 ATGGCTGAAGCATCCTCATTTCTTGCACAGAAAAGGTATGCGGTCGTGACAGGAGCAAACAAAGGACTAGGACTAGAAATATGCGGACAGCTTGCTTCACAGGGGGTGACGGTACTGCTGACATCCAGAGATGAAAAACGAGGCTTAGAAGCCATTGAGGAGCTTAAGAAATCGGGGATTAATTCGGAAAATCTTGAATATCATCAGCTGGATGTTACTAAGCCAGCTAGTTTCGCTTCTCTGGCCGATTTCATCAAGGCCAAATTTGGCAAGCTTGATATCCTGGTGAACAATGCAGGGATCAGCGGTGTTATTGTAGATTATGCAGCTTTAATGGAAGCCATTCGCCGTCGAGGGGCAGAGATCAATTACGATGGAGTGATGAAACAGACCTACGAGCTAGCAGAGGAATGCTTGCAAACAAATTACTATGGTGTGAAAAGAACCATTAATGCTCTCCTTCCGCTACTTCAGTTTTCCGATTCACCAAGGATCGTCAATGTTTCCTCCGATGTTGGCCTCCTTAAGAAAATACCCGGCGAGAGAATCAGAGAAGCCTTAGGCGACGTGGAAAAACTTACGGAAGAAAGCGTGGACGGGATTTTAGACGAGTTTCTAAGAGATTTCAAGGAAGGCAAGATCGCAGAGAAAGGTTGGCCTACGTTTAAGAGCGCCTATTCAATCTCAAAGGCGGCGCTCAATTCGTACACGAGGGTTTTAGCACGGAAATACCCGTCGATCATCATCAACTGTGTCTGCCCGGGTGTCGTCAAAACCGATATCAATCTTAAAATGGGCCACTTGACGGTTGAAGAAGGCGCGGCCAGTCCCGTGAGGTTAGCACTCATGCCCCTTGGTTCGCCTTCCGGCCTGTTCTATACTCGAAACGAAGTAACTCCATTTGAATGA

[0251] SEQ ID NO:52 - Oxidoreductase enzyme (SoFSL-1) capable of enhancing the activity of fucosyltransferase MAEASSFLAQKRYAVVTGANKGLGLEICGQLASQGVTVLLTSRDEKRGLEAIEELKKSGINSENLEYHQLDVTKPASFASLADFIKAKFGKLDILVNNAGISGVIVDYAALMEAIRRRGAEINYDGVMKQTYELAEECLQTNYYGVKRTINALLPLLQFSDSPRIVNVSSDVGLLKKIPGERIREALGDVEKLTEESVDGILDEFLRDFKEGKIAEKGWPTFKSAYSISKAALNSYTRVLARKYPSIIINCVCPGVVKTDINLKMGHLTVEEGAASPVRLALMPLGSPSGLFYTRNEVTPFE *

[0252] SEQ ID NO:53 - Nucleic acid sequence encoding the SpolFSL enzyme according to SEQ ID NO:54. SpolFSL ATGGCTGAACAATCCAACTTTCTGGCTGAAAAAAGGTATGCAGTAGTGACAGGTGCAAACAAAGGAATAGGGCTTGAAATATGCAGACAGCTTGCTTCTCAAGGTGTGATTGTACTTATCACTTCTAGAGATGGAAAGAAAGGATTAGAAGCCCTTAATGATCTCATTAAATCTGGAATTAGCTCTGATAATCTTCATTATCATCAGCTTGATGTTACTGACCCTATGAGTATTACTGCTCTTGCTGGTTTCATCAATTCCAAATTTGGCAAGCTTGATATTCTGGTGAACAATGCTGGGATAGGTGGATTTATAATTGACTACGATGCTATCAAAGCAATAGGTTTTCGCAATATCAATTATGACGAGATGATGACACAAACATATGAGCTTGCAAAAGAATGCTTGGAAACAAACTACTATGGAGTTAAGAGAACAACTGAAGCTTTGCTTCCTCAGCTGGAGTTATCGGATTCACCAAGGATCATCAATGTCTCCTCTTCTACGGGGATGTTGAAGAATATACCAAATGAGAGGATCAGAGGAGTCTTGGGTGATGCAGAGAATCTTACAGAAGAAAAAGTTGAAGCGATTTTGAATGAGTTACTGACAGATTTCAAAGATGGTTCATTCAAAGAGAAAGAATGGCCTTCTAGAATGGCAGCTTATACACTGTCAAAGGCGGCTTTGAATGCATATGCAAGAATATTGGCTAAGAAATACCCGTCAATTATCATCAGTTGTGTTTGTCCTGGTGTTACTAAGACAGATATGAACGGAAACTTGGGACAATTAACAGTTGAAGAAGGGGCCGCAAGTCCGGTGAGAGTAGCATTGATGCCTCATGGTTCACCTTCCGGTCTTTTCTATGCAAGAAGCGAAGTTTCTTCATATGAATAA

[0253] SEQ ID NO: 54 - Oxidoreductase enzyme (SpolFSL) capable of enhancing the activity of fucosyltransferase MAEQSNFLAEKRYAVVTGANKGIGLEICRQLASQGVIVLITSRDGKKGLEALNDLIKSGISSDNLHYHQLDVTDPMSITALAGFINSKFGKLDILVNNAGIGGFIIDYDAIKAIGFRNINYDEMMTQTYELAKECLETNYYGVKRTTEALLPQLELSDSPRIINVSSSTGMLKNIPNERIRGVLGDAENLTEEKVEAILNELLTDFKDGSFKEKEWPSRMAAYTLSKAALNAYARILAKKYPSIIISCVCPGVTKTDMNGNLGQLTVEEGAASPVRVALMPHGSPSGLFYARSEVSSYE *

[0254] A nucleic acid sequence encoding an enzyme according to SEQ ID NO: 55 - SEQ ID NO: 56. *

[0255] Accession No. 56 - QA-Tri(X / A)-F * A glucosyltransferase (Qs-7-GlcT) capable of transferring a glucose residue to the C-3 position of the C-28 rhamnose residue of the derivative. MADRVINSYKKLHVVLFPWLAFGHMIPFLELAKLIAQKGHKISYISTPRNIRRLPKIPSHLSNNLNFIEFPLPHIPNLHENVESTNDVTHDNPIAYYLLIKALEGLQQPITTFLETSDPDWIIHDVFPQWITATASRLRISHAFYTTSSALRTASNYSPLMSSELPQDIATDFTTKSTNLLAAKVLVIRSCLELEPKEFEQCKNLCVSKTVIPLGVVPPSIQVNDNISNINDDDNDWVKIVEWLNQGKEKGSVIYVALGSEVSLSEQDLKEFALGLELSGLSFFWVFRNTGSYGLPAGFEDRVKGRGIVWTSWAPQVSILGHESIGGFLSHGGWSSVIESLSFGIPLVVFPFGADQGINAKQLEGKNAGVEIPRSEGTGSFTRKSVADLLRLVVVEEEGKVYRDGAKELRKLFGDKDLNHKYIDNFVKYMEEHITNAAN

[0256] A nucleic acid sequence encoding an enzyme according to Accession No. 57 - Accession No. 58.

[0257] Array number 58 - QA-Tri (X / A)-F * Rhamnosyltransferase enzyme (Qs-7-RhT) capable of transferring a rhamnose residue to the C-3 position of D-fucose in the C-28 chain of the derivative MTSNNSQLHIFFLPALSPGHMIPVIEMAKLVASRGVMATIVTTAHNLAFVSRTISTYSTKIKIVTIKFPYAEVGLPEGCEIIDSDTPPDILFRVIKALRLLQEPMEQLLSSYQPDCLVADAFFSWATNSAAKFNIPRLVFHVACLFSLCASHSIELYEPQKKVSSDSETFIIPSLPGEIKLTKMQLPADLPKTGVEAEYINKMVKAVHESVENSYGFIINSFYELEKDYVDYYRNVIGRKAWHIGPLSLCHADNIEEKSQRGKESSIAENECLKWLDSRKPDSVVYVGFGSLVNFSDSQLMEIALGLEASEKQFIWVVKKSKRNEQEKEEWLPEGFEKRTVGKGLIIRGWAPQLLIMDHEAVGGFVTHCGWNSTLEGVCGGVVMATWPVSYEQIYTEKLVTDVLKIGVSVGAQTCDGIVGGIIKSEAIEKAVNRIMEGIEAEEMRSRAKAFAKKARQSVKEGGSSYSDLNSLIEELSLKSLKH

[0258] Nucleic acid sequence encoding an enzyme according to Array number 59 - Array number 60 *

[0259] Sequence number 60 - QA-Tri (X / A)-F * Acetyltransferase enzyme (Qs-7-AcetylT) capable of transferring acetyl to the C-4 position of D-fucose in the C-28 chain of the derivative MKIETISTNCIKPSKPTPSHLRNIKLSDQHQHSPDVHSNFTFFYQSNQIDDAVVTVPSAAIDVAPATDAAIDVAAATDTAIDGAATNFSVQSKILHNCLATTLTSFYPIAGRFQNGDTIICNDEGAFFIEAKTDINMSNFLGHPDLLTVIREHLVPDATNPDYNGSILLLKFTLFGCGSTAITISMSHKIADLVTFITLLNCWTALARAGGGGGIGGGSDGFIPPDLNFLGQIVPDSDPSPKSATPEFFRNKKFVTKRFVFSASKIKEVKDKVMKEIRKQEDDIFPSRVDVVLALIWRSTLSSLSGSSGKFKPAIFMQAANLRTRTDPPLPETSIGNLVILFPLVVEKETDIELHELVNKLLDAKAWVNKLKKKFQGYDGGNDPLQVVEAIRCEALKEMGNVWKKSKDFSMYISSSFCNFLMNEVDFGWGKPVWVTNTPRTIMANTIYLLDTKEVGGVDALVQFEEEEITKLELNQELLQFATVNPIPIVI

[0260] Nucleic acid sequence encoding an enzyme according to sequence numbers 61 - 62

[0261] Accession No. 62 - QA-Tri(X / A)-F * Acetyltransferase enzyme (SOAP10) capable of transferring acetyl to the C-4 position of the D-fucose residue on the skeleton MGEVNHEEVEIEIISIETIKPSSLLPPKTPPKTITLSHLDQAAPLYYYPLLLYYTNTTTTTPTSQIRVDITSTLKTSLSKTLDKFHPIAGRCVDDSTICCNHQGIPFIETKVDSNILDVMNSPEKMKLLIKFLPHAEFQDVTRPVSDLNHLAFQVNVFRCGGVIIGSYVLHKLLDGISLGTFFKNWSTIANDERVKDDDLVQPDFEATIKAFPPRTATPMLPRNQQLPKAAEKPNNNPVKVLVTKSFVFDIVSLKKMMFMAKSELVPKPTKFETVTGFIWEQTLSTLRNSGVEVEHTSLIIPVNIRPRMSPPLPRGSMGNLLKNAKAQANTSSSNGLQDLVKEIHSSLSQTTQKINTPPPPPPPPTTTATTIHSSLSQTTQKINTPPPTTTTIHSSLSQTTQKINTTTTTTAEVILTKRKVDNPVTQNREGNYLFTSWCKIGLDEADFGFGKPVWVIPNDGRPPKVRNMIFLTDYRHPETGVEGIAAWITLEEKQMQCLKSNPEFLAFATPN

[0262] Nucleic acid sequence encoding an enzyme according to Accession No. 63 - Accession No. 64

[0263] Array number 64 - QA-tri (X / A)-F * An acetyltransferase enzyme (DMOT9) capable of transferring acetyl to the C-4 position of the D-fucose residue on the skeleton MMEVHTTSENCIKPSQPTPSHLQNLKLSNHHSQAPDIRTNLTFFFSSNFNNPVQPGDHDATTNFTLQSKLVQNSLATTLTILYPFAGRFRNDDTIICKDDGAFFIEAKTDTKLSDFLAQPDLPLAIMDKLVPVATDAKYNGSLLILKFTLFGCGGSAVTISITHKISDLATILTLLNCWTALSRGGDGGGSSPFIQPDLNFIGRPVPSTSEVPPPSSGKNFIPPNSKYVTKRFIFSAAKIKELKARVINKIRKEEDNVFPSRVDVVLALIWKCALASVNSGSRSGNAQTFRPSVMMQAVNLRNRTDPPLPESSIGNLAILLPVWVEKEEDTELHELVSRLLTVKVRANRLKKKYQGYEDPEQVIISMESDSVKEIIEVRKKLKDFSTYVAASVVNAPLYDVDFGWGKPAWVTSTPNTVMANSIYLLDTKDAGGIEVLMNMLKEEDMIVFESNQELLQSAMVNPTII

Claims

1. QA-Tri(X / R)-F * a method for producing -GR-Ac, wherein the acetyl (Ac) moiety is F * The rhamnose (R) residue is attached to the C-4 position of the D-fucose of F * is attached to the C-3 position of D-fucose, and the glucose (G) residue is F * and the method comprises attaching QA-tri(X / R)-F * of i. the enzyme queratic acid 28-O-fucoside[1,2]-rhamnoside[1,3] glucosyltransferase (QS-7-GlcT) having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. one or more enzymes selected from the enzyme queratic acid 28-O-fucoside[1,4]acetyltransferase (QS-7-AcetylT) having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60; the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62; or the enzyme (3S,5S,6S)-3,5-dihydroxy-6-methyloctanoyl-CoA transferase 9 (DMOT9) having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64; and iii. The enzyme queratic acid 28-O-fucoside[1,3]rhamnosyltransferase (QS-7-RhaT) having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58; In combination with QA-Tri(X / R)-F * - forming GR-Ac.

2. a) First, QA-Tri(X / R)-F * in combination with the enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 56, to produce QA-tri(X / R)-F * -G is formed; b) QA-Tri(X / R)-F * QA-tri(X / R)-G in combination with one or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60; the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62; or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, to produce QA-tri(X / R)-F. * -G-Ac is formed; c) QA-Tri(X / R)-F * QA-tri(X / R)-F is prepared by combining QA-tri(X / R)-G-Ac with the enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO:

58. * -Forming GR-Ac, The method of claim 1.

3. In steps a), b) and c), F * The method of claim 2, wherein is FRX.

4. a) First, QA-Tri(X / R)-F * in combination with one or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, to produce QA-tri(X / R)-F * -Ac; b) QA-Tri(X / R)-F * QA-tri(X / R)-F is synthesized by combining QA-tri(X / R)-Ac with the enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO:

58. * -R-Ac; c) QA-Tri(X / R)-F * QA-tri(X / R)-F is prepared by combining QS-7-GlcT with the enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO:

56. * -Forming GR-Ac, The method of claim 1.

5. In steps a) and b), F * is FR and in step c) F * The method of claim 4, wherein is FRX.

6. First, QA-Tri(X / R)-F * in combination with one or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, to produce QA-tri(X / R)-F * -Ac; b) QA-Tri(X / R)-F * QA-tri(X / R)-F is prepared by combining QS-7-GlcT with the enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO:

56. * -G-Ac is formed; c) QA-Tri(X / R)-F * QA-tri(X / R)-F is prepared by combining QA-tri(X / R)-G-Ac with the enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence with at least 70% sequence identity to SEQ ID NO:

58. * -Forming GR-Ac, The method of claim 1.

7. In step a), F * is FR and in steps b) and c) F * The method of claim 6, wherein is FRX.

8. Tri(X / R) is TriX and F * The method of claim 1, wherein is FRXA.

9. Biosynthesis of QA-tri(X / R)-F in the host * - a method for producing GR-Ac, the method comprising the steps of: To the host a) QA-Tri RF * and / or QA-Tri XF * expressing genes necessary for the biosynthesis of b) introducing a polynucleotide encoding: i. the enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. one or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. The enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

58.

10. Biosynthesized QA-triXF in the host * - a method for producing GR-Ac, the method comprising the steps of: To the host a) QA-Tri XF * expressing genes necessary for the biosynthesis of b) introducing a polynucleotide encoding: i. the enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. one or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. The enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

58.

11. F * The method of claim 10, wherein is FRXA.

12. Biosynthesized QA-triRF in the host * - a method for producing GR-Ac, the method comprising the steps of: To the host a) QA-Tri RF * expressing genes necessary for the biosynthesis of b) introducing a polynucleotide encoding: i. the enzyme QS-7-GlcT having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; ii. one or more enzymes selected from the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO: 60 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 60, the enzyme SOAP10 having the amino acid sequence of SEQ ID NO: 62 or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 62, or the enzyme DMOT9 having the amino acid sequence of SEQ ID NO: 64 or an enzyme having an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 64, and iii. The enzyme QS-7-RhaT having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

58.

13. Amino acid SEQ ID NO:60 is encoded by polynucleotide SEQ ID NO:59; Amino acid SEQ ID NO:58 is encoded by polynucleotide SEQ ID NO:57; Amino acid SEQ ID NO:56 is encoded by polynucleotide SEQ ID NO:55; Amino acid SEQ ID NO:62 is encoded by polynucleotide SEQ ID NO:61, and amino acid SEQ ID NO:64 is encoded by polynucleotide SEQ ID NO:

63. The method according to any one of claims 9 to 12.

14. a glucosyltransferase enzyme (QS-7-GlcT) having the amino acid sequence of SEQ ID NO: 56, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 56; A rhamnosyltransferase enzyme (QS-7-RhaT) having the amino acid sequence of SEQ ID NO: 58, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 58; or An acetyltransferase enzyme (QS-7-AcetylT) having the amino acid sequence of SEQ ID NO:60, or an enzyme having an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

60.

15. A polynucleotide encoding any one of the enzymes of claim 14.

16. A vector comprising the polynucleotide of claim 15.

17. A host cell comprising the polynucleotide of claim 15.

18. A host cell transformed with the vector of claim 16.

19. 19. The host cell of claim 17 or 18, wherein the host cell is a plant cell or a microbial cell.

20. 20. A plant or microbial biological system comprising the host cell of claim 19.

21. 21. The biological system of claim 20, wherein the biological system is yeast or Nicotiana benthamiana.

22. The method comprises: * The method of any one of claims 1 to 12, further comprising isolating the -GR-Ac derivative.

23. QA-tri(X / R)-F obtained by the method according to claim 22 * -GR-Ac derivative.

24. 24. The derivative of claim 23, wherein the derivative is 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucopyranosiduronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-chiric acid (QA-triX-FRXA-GR-Ac) acetylated at the C-4 position of the D-fucose of the core C-28 chain with a rhamnose moiety attached to the C-3 position of the D-fucose of the C-28 chain and a glucose moiety attached to the C-3 position of the core C-28 rhamnose moiety.

25. QA-tri(X / R)-F according to claim 23 as an adjuvant * -Use of GR-Ac derivatives.

26. 26. The use according to claim 25, wherein the adjuvant is a liposome preparation or an immunostimulating complex (ISCOM) preparation.

27. 25. The ISCOM formulation of claim 24, wherein the ISCOM formulation is QA-Tri(X / R)-F. * 27. The use of claim 26, comprising a first ISCOM matrix containing the -GR-Ac derivative and a second ISCOM matrix.

28. 26. The use of claim 25, wherein the adjuvant further comprises a TLR4 agonist.

29. 29. The use according to claim 28, wherein the TLR4 agonist is 3D-MPL.

30. QA-tri(X / R)-F according to claim 23 * -An adjuvant composition comprising a GR-Ac derivative.

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