Methods and compositions relating to synthesis of QS-7 molecules

Abstract

The present invention relates to biosynthetic pathways of QS-7 molecule precursors, as well as pathways for making QS-7 molecules, enzymes involved, products produced, and uses of the products.

Description

[0001] The present invention relates to the biosynthetic pathway of QS-7 molecule precursors, as well as the pathway for producing QS-7 molecules, the enzymes involved, the products produced and the uses of the products. Background Art

[0002] QS-7 is a natural saponin extract extracted from the bark of the Chilean soap bark tree (Quillaja saponaria). The QS-7 extract was originally identified as a purified fraction of the crude bark extract of the Molina soap bark tree (Quillaja Saponaria Molina) obtained by RP-HPLC purification (peak 7) (Kensil et al. 1991). The QS-7 molecule contains a central triterpene core skeleton (saponic acid) to which branched trisaccharides are attached at the triterpene C-3 oxygen functional group, and the sugar chains are connected to the triterpene C-28 carboxylate group. The difference between QS-7 and QS-21 is the structure of the sugar chain at the C-28 position (see Figure 1 ). The QS-21 structure shows 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, in which the terminal sugar is apiose, and to which two additional sugars are incorporated (thus generating 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. Another difference between the two is that QS-7 incorporates an acetyl moiety at the C-4 position of the fucose residue, instead of incorporating an acyl chain on the fucose residue (QS-21) (see Figure 1 ).

[0003] For many years, it has been known that saponins in Quillaja, including QS-7, have potent immunostimulatory properties, capable of enhancing antibody production and specific T-cell responses. QS-7 exhibits similar potency to QS-21 and has reduced toxicity (Kensil et al. 1991). These properties have led to the development of vaccine adjuvants based on Quillaja saponins. Of particular note, QS-7 is present in Novavax's "Matrix-M" (as part of a saponin fraction named "Fraction A" - see, e.g., WO2017 / 161151), used in the NVX-CoV2373 COVID-19 vaccine.

[0004] The present invention describes methods for synthesizing QS-7 molecule precursors, QS-7 molecules themselves and variants thereof, without purification from the natural Quillaja plant. The present invention also describes the resulting products, which can be used as adjuvants in vaccine formulations. The present invention also relates to methods, vectors, host cells and biological systems for producing the products. Summary of the invention

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

[0006] For simplicity, the linear tetrasaccharide sugar chain at C-28 of QA and its precursors (i.e., a sugar chain with only two or three sugars at C-28) will be referred to as F* in the remainder of this specification. Therefore, in the sense of the present invention, "F*" should be understood as FR, FRX, FRXA and / or FRXX (for further simplification, FRXA and FRXX can also be named FRX(X / A)) (see the list of abbreviations herein).

[0007] The present invention includes biosynthetic preparations of QA-Tri(X / R)-F*-GR-Ac and precursors thereof. The present invention also relates to the use of QA-Tri(X / R)-F*-GR-Ac, such as the QS-7 molecule (QA-TriX-FRXA-GR-Ac), and precursors and variants thereof, such as as adjuvants.

[0008] QA synthesis

[0009] QA is derived from the simple triterpene β-amyrin, which is synthesized by cyclization of the universal linear precursor 2,3-oxidosqualene (OS) under the action of oxidosqualene cyclase (OSC). This biosynthesis is known in the art, for example in WO2019 / 122259, the contents of which are incorporated by reference. The β-amyrin scaffold is further oxidized with carboxylic acids, alcohols and aldehydes at C-28, C-16α and C-23, respectively, under the action of a series of three cytochrome P450 monooxygenases to form saponin (QA). OSC and C-28, C16α and C-23 oxidases are referred to herein as QsbAS (β-amyrin synthase), QsCYP716-C-28, QsCYP716-C-16α and QsCYP714-C-23 oxidases, respectively. Figure 2 The biosynthetic pathway is given.

[0010] Synthesis of C-3 branched trisaccharide

[0011] The C-3 branched trisaccharide chain starts with a D-pyranoglucuronic acid (D-GlcpA) residue attached to the C-3 position of the QA backbone via a β-bond. Two sugars are attached to the D-GlcpA residue: a D-galactopyranose (D-Galp) residue attached via a β-1,2-bond, and a D-xylopyranose (D-Xylp) moiety or an L-rhamnose (L-Rhap) residue attached via a β-1,3-bond or α-1,3-bond, respectively. Figure 3 Schematic diagram of the glycosylation of QA to 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucuronopyranose}-saponic acid (QA-TriR) or 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-glucuronopyranose}-saponic acid (QA-TriX) is shown.

[0012] Seven enzymes with activities associated with the production of QA 3-O trisaccharides (QA-TriX or QA-TriR) have been identified, for example in WO2020 / 260475, the contents of which are incorporated by reference. These include: two functionally redundant glucuronyl transferases, CSL1 and CslG2, which can add an initial β-D-pyranoglucuronic acid moiety at the C-3 position of saponin; a galactosyltransferase, Qs-3-O-GalT, which adds a β-D-galactopyranose residue to the C-2 position of β-D-pyranoglucuronic acid; a xylosyltransferase, Qs_0283870, which adds a β-D-xylopyranose residue to the C-2 position of β-D-pyranoglucuronic acid; and a β-D-xylose residue to the C-3 position of β-D-pyranoglucuronic acid. glucuronic acid at the C-3 position; two rhamnosyltransferases, DN20529_c0_g2_i8 and Qs_0283850, which add α-L-rhamnose pyranose residues to the C-3 position of β-D-glucuronic acid; and the bifunctional enzyme Qs-3-O-RhaT / XylT, which can add either β-D-xylopyranose residues or α-L-rhamnose pyranose residues to the C-3 position of β-D-glucuronic acid (see Figure 3 ).

[0013] For simplicity, throughout this application, QA derivatives comprising a branched trisaccharide at position C-3 may be designated as "QA-TriX", "QA-TriR" or "QA-Tri(X / R)" (see list of abbreviations herein).

[0014] Synthesis of C-28 Straight-chain Tetrasaccharide

[0015] F* is initiated by attaching a D-fucose residue to the C-28 position of the QA backbone via a β-bond. This step is followed by attaching an L-rhamnose residue to the C-2 position of the fucose residue via an α-bond, and then attaching a D-xylose residue to the C-4 position of the rhamnose residue via a β-bond. Finally, a D-xylose residue or a D-apiose residue is attached to the C-3 position of the xylose residue via a β-bond.

[0016] Ten enzymes with activities associated with F* production have been identified, as reported in PCT / EP2021 / 087323. These include: Qs-28-O-FucT (SEQ ID NO 2), which transfers a D-fucose residue to the C-28 position of the QA backbone via a β-bond; Qs-28-O-RhaT (SEQ ID NO 4), which transfers an L-rhamnose residue to a D-fucose moiety; Qs-28-O-XylT3 (SEQ ID NO 6), which transfers a D-xylose moiety to an L-rhamnose residue; Qs-28-O-XylT4 (SEQ ID NO 8), which attaches a β-D-xylose residue to a β-D-xylose residue; Qs-28-O-ApiT4 (SEQ ID NO 10), which attaches a β-D-apiose residue to a β-D-xylose residue ( Figure 4 ). The oxidoreductase QsFucSyn (SEQ ID No. 12), which increases the production of UDP-D-fucose and / or reduces the 4-keto group of 4-keto-6-deoxyglucose when added to the QA backbone, and QsFucSyn-Like enzymes, such as QsFSL-1 (SEQ ID No. 48), QsFSL-2 (SEQ ID No 50) or SoFSL-1 (SEQ ID No 52), were also identified as having activities associated with F* production. A UDP-Apiose / UDP-Xylose Synthase QsAXS1 (SEQ ID NO 14) was also previously identified that enhances the activity of celery glycosyltransferase by increasing the availability of UDP-α-D-celeryose.

[0017] Synthesis of C-28 acetylated branched hexasaccharide

[0018] The present invention describes for the first time a biosynthetic pathway for adding a glucose residue at the C-3 position of the rhamnose residue of F*, adding a rhamnose residue at the C-3 position of the D-fucose residue of F*, and adding an acetyl moiety at the C-4 position of the D-fucose residue of F* to form the QS-7 molecule and its precursors and variants. As a result, the QS-7 molecule contains a branched hexasaccharide chain at the C-28 position and an acetyl moiety at the C-4 position of the D-fucose residue of F* (see Figure 1 ).

[0019] Therefore, the present invention provides a method for producing QS-7 and its precursors and variants. Also provided are an enzyme used in the method, a polynucleotide encoding the enzyme, a vector comprising the polynucleotide, a host cell transformed with the vector, and the use of QS-7 molecules, its precursors and variants as adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The structures of QS-7 and QS-21 are shown. Both have a backbone formed by the triterpenoid saponin (QA). The C-3 position of QA is characterized by a branched trisaccharide consisting of β-D-pyranoglucuronic acid (D-GlcpA), β-D-galactopyranose (D-Galp), and β-D-pyranose xyloylose (D-xylp). The C-28 position is characterized by a linear sugar chain consisting of β-D-pyranose fucose (D-fucp), α-L-pyranose rhamnose, β-D-pyranose xyloylose, and a terminal β-D-apifuranose (D-apif) (for QS-21 and QS-7) or β-D-pyranose xyloylose (for QS-21). In QS-7, a glucose residue is incorporated at the C-3 position of the rhamnose residue in the linear sugar chain at the C-28 position, and a rhamnose residue is incorporated at the C-3 position of the D-fucose in the linear sugar chain at the C-28 position. D-fucose is also characterized by having an acetyl moiety at the C-4 position.

[0021] Figure 2 The production of squalene (QA) from 2,3-oxidized squalene via β-amyrin is shown. The pathway starting from β-amyrin requires oxidation at three positions (C-28, C-23, and C-16α). For simplicity, these oxidation steps are shown in a linear fashion; however, they can occur in any order.

[0022] Figure 3The process of generating QA-TriR or QA-TriX from saponic acid (QA) is shown. β-D-pyranoglucuronic acid (β-D-GlcpA) is added to the C-3 position of QA by glucuronyltransferases QsCLS1 or QsCslG2 to form QA-Mono. β-D-galactopyranose (β-D-Galp) is added to the C-2 position of pyranoglucuronic acid by galactosyltransferase Qs-3-O-GalT to form QA-Di. α-L-pyranoglucuronic acid (α-L-Rhap) can be attached to the C-3 position of pyranoglucuronic acid by monofunctional rhamnosyltransferases DN20529_c0_g2_i8 or Qs_0283850, or by bifunctional Qs-3-O-RhaT / XylT to form QA-TriR. Alternatively, β-D-xylopyranose (β-D-Xylp) can be attached to the C-3 position of glucopyranose by the monofunctional xylosyltransferase Qs_0283870 or by the bifunctional Qs-3-O-RhaT / XylT to form QA-TriX.

[0023] Figure 4 The process of producing QA-Tri(X / R)-FRX(X / A) from QA-Tri(X / R) is shown. The chain starts with β-D-fucopyranose (β-D-Fucp) attached to C-28 of QA via an ester bond, followed by α-1,2-L-rhamnose (α-L-Rhap) and β-1,4-D-xylopyranose (β-D-Xylp). The terminal sugar of the chain can be β-1,3-D-xylopyranose (β-D-Xylp) or β-1,3-D-apiofuranosyl (β-D-Apif). For simplicity, the resulting QA derivative can be named QA-Tri(X / R)-FRX(X / A).

[0024] Figure 5 The production of QA-TriX-FRXA-G in Nicotiana benthamiana is shown. The gene set (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / Csl1 / C3-GalT / C3-XylT / QsFucSyn / C-28-FucT / C28-RhaT / C28-XylT3 / C28-ApiT4) that produces the QA-TriX-FRXA product was transiently expressed in N. benthamiana together with UGT-BI. LC-MS analysis of leaf extracts revealed the presence of a product with a mass consistent with the addition of hexose residues, which is expected to be glucose. The new product was named QA-TriX-FRXA glucoside (QA-TriX-FRXA-G).

[0025] Figure 6 The production of QA-TriX-FRXA-Ac in Nicotiana benthamiana is shown. The gene set (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / Csl1 / C3-GalT / C3-XylT / QsFucSyn / C-28-FucT / C28-RhaT / C28-XylT3 / C28-ApiT4) that produces the QA-TriX-FRXA product was transiently expressed in Nicotiana benthamiana along with ACT-19'. LC-MS analysis of leaf extracts revealed the presence of a product with a mass consistent with the addition of an acetyl group. The new product was named QA-TriX-FRXA Acetyl (QA-TriX-FRXA-Ac).

[0026] Figure 7 The production of QA-TriX-FRXA-R-Ac in Nicotiana benthamiana is shown. The gene set (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / Csl1 / C3-GalT / C3-XylT / QsFucSyn / C-28-FucT / C28-RhaT / C28-XylT3 / C28-ApiT4 / ACT-19') that produces the QA-TriX-FRXA product was transiently expressed in Nicotiana benthamiana along with UGT-0023500. LC-MS analysis of leaf extracts revealed the presence of a product with a mass consistent with the addition of deoxyhexoses, expected to be rhamnose. The new product was named QA-TriX-FRXA-Ac rhamnoside (QA-TriX-FRXA-R-Ac).

[0027] Figure 8 The production of QS-7 (QA-TriX-FRXA-GR-Ac) in Nicotiana benthamiana is shown. The gene set (tHMGR / QsbAS / CYP716-C-28 / CYP716-C-16α / CYP714-C23 / Csl1 / C3-GalT / C3-XylT / C-28-FucT / C28-RhaT / C28-XylT3 / C28-ApiT4) that produces the QA-TriX-FRXA product was transiently expressed in Nicotiana benthamiana along with the GlcT, RhaT and AcetylT genes required to convert this precursor to QS-7. LC-MS analysis of leaf extracts expressing all QS-7 genes revealed the presence of a product with the same retention time and mass as the authentic QS-7 standard. This peak was absent in control samples lacking one of the GlcT, RhaT or AcetylT.

[0028] Fig. 9The quercetin (QA) triterpene core of semi-purified QS-7 (QA-TriX-FRXA-GR-Ac) in MeOH-d4 (600, 150 MHz) is shown. 1 H. 13 C spectral data.

[0029] Fig.10 The semi-purified QS-7 (QA-TriX-FRXA-GR-Ac) shows C3, C 28 Oligosaccharides in MeOH-d4 (600, 150 MHz) 1 H. 13 C spectral data. DETAILED DESCRIPTION

[0030] A first aspect of the invention is a method for making QA-Tri(X / R)-F*-GR-Ac, wherein an acetyl (Ac) moiety is attached to the C-4 position of the D-fucose of F*, a rhamnose (R) residue is attached to the C-3 position of the D-fucose of F*, and a glucose (G) residue is attached to the C-3 position of the rhamnose residue of F*. The method comprises combining QA-Tri(X / R)-F* with:

[0031] i. an enzyme having the amino acid sequence of SEQ ID NO 56, sapote 28-O-fucoside [1,2]-rhamnoside [1,3] glucosyltransferase (QS-7-GlcT), or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0032] ii. one or more enzymes selected from the group consisting of: an enzyme having an amino acid sequence of SEQ ID NO 60, saponic acid 28-O-fucoside [1,4] acetyltransferase (QS-7-AcetylT), or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme having an amino acid sequence of SEQ ID NO 62, SOAP10, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme having an amino acid sequence of SEQ ID NO 64, (3S,5S,6S)-3,5-dihydroxy-6-methyloctanoyl-CoA transferase 9 (DMOT9), or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and

[0033] iii. an enzyme having the amino acid sequence of SEQ ID NO 58, sapote 28-O-fucoside [1,3] rhamnosyltransferase (QS-7-RhaT), or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 58.

[0034] In this aspect of the invention, QA is quince acid;

[0035] Tri(X / R) is a branched trisaccharide located at the C-3 position of the QA backbone, which terminates with a xylose residue (X) or a rhamnose residue (R);

[0036] F* is: a disaccharide of a β-D-fucose residue (F) and R, also known as FR; a trisaccharide of F, R and X, also known as FRX; a tetrasaccharide of F, R, X and X, also known as FRXX, or a tetrasaccharide of F, R, X and a β-D-apiose residue (A), also known as FRXX;

[0037] G is a glucose residue; and

[0038] Ac is an acetyl moiety.

[0039] A second aspect of the present invention is a method for producing biosynthetic QA-Tri(X / R)-F*-GR-Ac in a host. The method comprises the following steps:

[0040] a) expressing genes required for the biosynthesis of QA-TriR-F* and / or QA-TriX-F*, and

[0041] b) introducing into the host a polynucleotide encoding:

[0042] i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0043] ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64, or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and

[0044] 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 with SEQ ID NO 58.

[0045] In this aspect of the invention, QA is quince acid;

[0046] Tri(X / R) is a branched trisaccharide located at the C-3 position of the QA backbone, which terminates with a xylose residue (X) or a rhamnose residue (R);

[0047] F* is: a disaccharide of a β-D-fucose residue (F) and R, also known as FR; a trisaccharide of F, R and X, also known as FRX; a tetrasaccharide of F, R, X and X, also known as FRXX; or a tetrasaccharide of F, R, X and a β-D-apiose residue (A), also known as FRXA;

[0048] G is a glucose residue; and

[0049] Ac is an acetyl moiety.

[0050] A third aspect of the present invention is a method for producing biosynthetic QA-TriX-F*-GR-Ac in a host. The method comprises the following steps:

[0051] a) express the genes required for the biosynthesis of QA-TriX-F*, and

[0052] b) introducing into the host a polynucleotide encoding:

[0053] i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0054] ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64, or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and

[0055] 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 with SEQ ID NO 58.

[0056] In this aspect of the invention, QA-TriX is 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-saponic acid;

[0057] F* is: a disaccharide of a β-D-fucose residue (F) and a rhamnose residue (R), also known as FR; a trisaccharide of F, R and a xylose residue (X), also known as FRX; a tetrasaccharide of F, R, X and X, also known as FRXX; or a tetrasaccharide of F, R, X and a β-D-apiose residue (A), also known as FRXA;

[0058] G is a glucose residue; and

[0059] Ac is an acetyl moiety.

[0060] In a third aspect of the invention, F* may be FRXA. Thus, the invention includes a method for producing biosynthesized QA-TriX-FRXA-GR-Ac in a host. The method comprises the following steps:

[0061] a) expressing genes required for the biosynthesis of QA-TriX-FRXA, and

[0062] b) introducing into the host a polynucleotide encoding:

[0063] i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0064] ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64, or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and

[0065] 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 with SEQ ID NO 58.

[0066] A fourth aspect of the present invention is a method for producing biosynthetic QA-TriR-F*-GR-Ac in a host. The method comprises the following steps:

[0067] a) express the genes required for the biosynthesis of QA-TriR-F*, and

[0068] b) introducing into the host a polynucleotide encoding:

[0069] i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0070] ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64, or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and

[0071] 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 with SEQ ID NO 58.

[0072] In this aspect of the invention, QA-TriR is 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-saponic acid;

[0073] F* is: a disaccharide of a β-D-fucose residue (F) and a rhamnose residue (R), also known as FR; a trisaccharide of F, R and a xylose residue (X), also known as FRX; a tetrasaccharide of F, R, X and X, also known as FRXX; a tetrasaccharide of F, R, X and a β-D-apiose residue (A), also known as FRXA;

[0074] G is a glucose residue; and

[0075] Ac is an acetyl moiety.

[0076] In the first aspect of the present invention, steps (i), (ii) and (iii) may occur sequentially. QA-Tri(X / R)-F* is first combined with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56, to form QA-Tri(X / R)-F*-G. In particular, F* may be FRX. F* may also be FRX(X / A). QA-Tri(X / R)-F*-G is then combined with one or more enzymes selected from the following to form QA-Tri(X / R)-F*-G-Ac: the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO 60 or an enzyme having an amino acid sequence with 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 with 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 with at least 25% sequence identity to SEQ ID NO 64. In particular, F* may be FRX. F* may also be FRX(X / A). Finally, QA-Tri(X / R)-F*-G-Ac is combined with an enzyme QS-7-RhaT having an amino acid sequence of SEQ ID NO 58 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 58 to form QA-Tri(X / R)-F*-GR-Ac. In particular, F* may be FRX. F* may also be FRX(X / A). In steps (i), (ii) and (iii), F* may be FRX.

[0077] The steps of this aspect of the invention may also be performed in the order of (ii), (iii) and then (i). First, QA-Tri(X / R)-F* is combined with one or more enzymes selected from the following to form QA-Tri(X / R)-F*-Ac: the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO 60 or an enzyme having an amino acid sequence with 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 with 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 with at least 25% sequence identity to SEQ ID NO 64. In particular, F* may be FR. F* may also be FRX. F* may also be FRX(X / A). Then, QA-Tri(X / R)-F*-Ac is combined with an enzyme QS-7-RhaT having an amino acid sequence of SEQ ID NO 58 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 58 to form QA-Tri(X / R)-F*-R-Ac. In particular, F* may be FR. F* may also be FRX. F* may also be FRX(X / A). Finally, QA-Tri(X / R)-F*-R-Ac is combined with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56 to form QA-Tri(X / R)-F*-GR-Ac. F* may be FRX. F* may also be FRX(X / A). In steps (i) and (ii), F* may be FR, and in step (iii), F* may be FRX.

[0078] The steps of this aspect of the invention may also be performed in the order of (ii), (i) and then (iii). First, QA-Tri(X / R)-F* is combined with one or more enzymes selected from the following to form QA-Tri(X / R)-F*-Ac: the enzyme QS-7-AcetylT having the amino acid sequence of SEQ ID NO 60 or an enzyme having an amino acid sequence with 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 with 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 with at least 25% sequence identity to SEQ ID NO 64. F* may be FR. F* may also be FRX. F* may also be FRX(X / A). Then, QA-Tri(X / R)-F*-Ac is combined with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56 to form QA-Tri(X / R)-F*-G-Ac. F* may be FRX. F* may also be FRX(X / A). Finally, QA-Tri(X / R)-F*-G-Ac is combined with an enzyme QS-7-RhaT having an amino acid sequence of SEQ ID NO 58 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 58 to form QA-Tri(X / R)-F*-GR-Ac. F* may be FRX. F* may also be FRX(X / A). In step (i), F* may be FR, and in steps (ii) and (iii), F* may be FRX.

[0079] In a first aspect of the invention, Tri(X / R) may be TriX and F* may be FRXA. Thus, the invention includes a method of making QA-TriX-FRXA-GR-Ac, wherein an acetyl (Ac) moiety is attached to the C-4 position of the D-fucose of FRXA, a rhamnose (R) residue is attached to the C-3 position of the D-fucose of FRXA, and a glucose (G) residue is attached to the C-3 position of the rhamnose residue of FRXA, wherein the method comprises combining QA-TriX-FRXA with:

[0080] i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0081] ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64, or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and

[0082] 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 with SEQ ID NO 58.

[0083] When Tri(X / R) is TriX and F* is FRXA, the steps of the first aspect of the invention may occur in the order of (i), (ii) and then (iii). These steps may also be performed in the order of (ii), (iii) and then (i). These steps may also be performed in the order of (ii), (i) and then (iii).

[0084] As defined herein, F* can be FR, FRX, FRXA and / or FRXX. The sugar of the F* chain is added at the C-28 position of QA-Tri (X / R). When F* is a mixture comprising FRXX and FRXA, the ratio of FRXX to FRXA can be different. The percentage ratio of FRXX to FRXA in the mixture can be different. Suitably, the mixture comprises 10 to 90% FRXX, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and 90 to 10% FRXA, such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%. Preferably, the mixture comprises 60% FRXX and 40% FRXA, or each comprises 50%.

[0085] In embodiments where the acetyl (Ac) moiety is attached to the C-4 position of the D-fucose of F*, F* is FR, FRX, FRXA and / or FRXX. In particular, F* may be FRXA. Similarly, in embodiments where QA-Tri(X / R)-F* is combined with one or more enzymes selected from the following to form QA-Tri(X / R)-F*-Ac, F* is FR, FRX, FRXA and / or FRXX: the enzyme is: QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; SOAP10 having an amino acid sequence of SEQ ID NO 62 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or DMOT9 having an amino acid sequence of SEQ ID NO 64 or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64. In particular, F* may be FRXA. In an embodiment where QA-Tri(X / R)-F*-G is combined with one or more enzymes selected from the following to form QA-Tri(X / R)-F*-G-Ac, F* is FRX, FRXA and / or FRXX, and in particular F* may be FRXA: the enzyme is: QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; SOAP10 having an amino acid sequence of SEQ ID NO 62 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or DMOT9 having an amino acid sequence of SEQ ID NO 64 or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64.

[0086] In embodiments where a rhamnose (R) residue is attached to the C-3 position of the D-fucose of F*, F* is FR, FRX, FRXA and / or FRXX, and the acetyl moiety must be attached to the C-4 position of the D-fucose of F*. In embodiments where QA-Tri(X / R)-F*-Ac is combined with an enzyme QS-7-RhaT having an amino acid sequence of SEQ ID NO 58 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 58 to form QA-Tri(X / R)-F*-R-Ac, F* is FR, FRX, FRXA and / or FRXX. In particular, F* may be FRXA. In the embodiment where QA-Tri(X / R)-F*-G-Ac is combined with 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 with SEQ ID NO 58 to form QA-Tri(X / R)-F*-GR-Ac, F* is FRX, FRXA and / or FRXX. In particular, F* can be FRXA.

[0087] In embodiments 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* may be FRXA. In embodiments where QA-Tri(X / R)-F* is combined with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56 to form QA-Tri(X / R)-F*-G, F* is FRX, FRXA and / or FRXX. In particular, F* may be FRXA. In embodiments where QA-Tri(X / R)-F*-Ac is combined with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56 to form QA-Tri(X / R)-F*-G-Ac, F* is FRX, FRXA and / or FRXX. In particular, F* may be FRXA. In an embodiment where QA-Tri(X / R)-F*-R-Ac is combined with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56 to form QA-Tri(X / R)-F*-GR-Ac, F* is FRX, FRXA and / or FRXX. In particular, F* may be FRXA.

[0088] 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.

[0089] When QA-Tri(X / R) is a mixture comprising QA-TriX and QA-TriR, the ratio of QA-TriX to QA-TriR may vary. The percentage ratio of QA-TriX to QA-TriR in the mixture may vary. Suitably, the mixture comprises 10 to 90% QA-TriX, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and 90 to 10% QA-TriR, such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.

[0090] Synthesis of C-28 acetylated branched hexasaccharide

[0091] The QS-7 molecule incorporates a glucose residue at the C-3 position of the rhamnose residue of F*, a rhamnose residue at the C-3 position of the D-fucose of F*, and an acetyl moiety at the C-4 position of the D-fucose of F*. The inventors have discovered enzymes that allow the addition of glucose residues, rhamnose residues, and acetyl moieties to desired positions in the core molecule in vitro and in vivo. "Core molecule" refers to one or more of the following QA derivatives: QA-TriX-FR, QA-TriX-FRX, QA-TriX-FRXX, QA-TriX-FRXA, QA-TriR-FR, QA-TriR-FRX, QA-TriR-FRXA, QA-TriR-FRXX, QA-Tri(X / R)-FR, QA-Tri(X / R)-FRX, QA-Tri(X / R)-FRXA, QA-Tri(X / R)-FRXX.

[0092] In the method of the present invention, the steps of adding glucose and rhamnose residues and acetyl moieties may be performed in a specific order or in any order or simultaneously.

[0093] In the method of the present invention, the transfer of the acyl moiety to the C-4 position of the D-fucose of F* (e.g., the transfer of the acyl moiety to QA-Tri(X / R)-F* to form QA-Tri(X / R)-F*-Ac) can be performed by the enzyme QS-7-AcetylT (SEQ ID NO 60) or an enzyme having at least 70% sequence identity with the QS-7-AcetylT sequence, 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 with 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 with SEQ ID NO 64. These enzymes are capable of transferring the acyl unit to the C-4 position of the D-fucose of the F* chain. The function of the enzyme can be determined, for example, according to the method described in Example 2.

[0094] The functionality of QS-7-AcetylT, SOAP10, or DMOT9 can be determined by expressing the enzymes required to produce QA-Tri(X / R)-F* and the QS-7-AcetylT, SOAP10, or DMOT9 candidate in a heterologous host such as N. benthamiana or yeast. The presence of the expected product can be assessed by LC-MS analysis, ultimately supplemented by NMR analysis. Alternatively, in vitro testing may be preferred, where QA-Tri(X / R)-F* is purified from a plant extract or is produced in vitro in an assay containing sapogenin and the glycosyltransferases necessary to produce QA-Tri(X / R)-F*, or β-amyrin and the enzymes necessary to produce QA-Tri(X / R)-F*. The activity of the candidate QS-7-AcetylT, SOAP10, or DMOT9 is then tested in vitro on the QA-Tri(X / R)-F* substrate, and product formation is determined by LC-MS analysis.

[0095] In the present specification, when an enzyme (SEQ ID NO) is mentioned, it refers to the enzyme according to that SEQ ID NO. For example, "QS-7-AcetylT (SEQ ID NO 60)" refers to the enzyme QS-7-AcetylT according to SEQ ID NO 60.

[0096] The enzyme used in the present invention may include one or more conservative amino acid substitutions so that the resulting enzyme has a similar amino acid sequence and / or retains the same function. It is known to those skilled in the art that various amino acids have similar biochemical properties and are therefore "conservative". One or more such amino acids of a protein (e.g., enzyme), polypeptide, or peptide may generally be replaced by one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

[0097] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine are generally interchangeable (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used interchangeably (because they have relatively short side chains), and valine, leucine, and isoleucine are preferably used interchangeably (because they have larger hydrophobic aliphatic side chains). Other amino acids that are generally interchangeable include: 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 performed using naturally occurring or non-naturally occurring amino acids. For example, the methyl group on alanine can be replaced with an ethyl group, and / or minor changes can be made to the peptide backbone. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present. Substitutions of this nature are often referred to as "conservative" amino acid substitutions.

[0098] As known in the art, "identity" is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences determined by comparing sequences. In the art, identity also means the degree of sequence correlation between polypeptide or polynucleotide sequences (as the case may be), as determined by the matching between the strings of such sequences. Although there are many methods for measuring the identity between two polypeptide sequences or two polynucleotide sequences, the method generally used to determine identity is encoded in a computer program. Preferred computer programs for determining the consistency between two sequences include but are not limited to GCG program packages ((Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN and FASTA (Atschul et al., J.Molec.Biol.215, 403 (1990)).。

[0099] Amino acid sequences can be compared using programs such as the CLUSTAL program. The program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. Amino acid identity or similarity (identity plus conservation of amino acid type) can be calculated for the optimal alignment. Programs such as BLASTx compare the longest stretch of similar sequences and assign values ​​to the fit. Thus, comparisons can be obtained when several similar regions are found, each with different scores.

[0100] The percent identity of two amino acid sequences or two polynucleotide sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence to optimally align with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. An "optimal alignment" is the alignment of two sequences that achieves the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the compared sequences (i.e., % identity = number of identical positions / total number of positions x 100).

[0101] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, which was modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The algorithm is included in the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleic acid searches can be performed using the NBLAST program (score=100, wordlength=12) to obtain nucleic acid sequences homologous to nucleic acid molecules. BLAST protein searches can be performed using the XBLAST program (score=50, wordlength=3) to obtain amino acid sequences homologous to protein molecules used in the present invention. To obtain gapped alignments for alignment purposes, GappedBLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search to detect long-distance relationships (Id.) between molecules. When using BLAST, GappedBLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm 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, has incorporated this algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10: 3-5; and FASTA as 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.

[0102] Any suitable method can be used to introduce mutations (including conservative substitutions, insertions and deletions) into the sequence, including but not limited to methods based on polymerase chain reaction (PCR), restriction enzyme cloning or ligation-independent cloning (LIC) steps. These methods are described in detail in many standard molecular biology texts. For more details on polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning-A Laboratory Manual (3rd Ed.) CSHL Press. More information on ligation-independent cloning (LIC) steps can be found in Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6.

[0103] In the method of the present invention, the transfer of the acyl moiety to the C-4 position of the D-fucose of F* can be performed 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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 56. Thus, in some embodiments, QS-7-AcetylT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 60, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of transferring an acyl unit to the C-4 position of the D-fucose of F*.

[0104] The transfer of the acyl moiety to the C-4 position of the D-fucose of F* can also be performed by the enzyme SOAP10 (SEQ ID NO 62) or an enzyme having at least 70% sequence identity to the SOAP10 (SEQ ID NO 62) sequence. The amino acid sequence of the SOAP10 enzyme may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 62. Thus, in some embodiments, SOAP10 is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 62, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of transferring the acyl unit to the C-4 position of the D-fucose of F*.

[0105] The transfer of the acyl moiety to the C-4 position of the D-fucose of F* can also be performed by the enzyme DMOT9 (SEQ ID NO 64) or an enzyme having at least 25% sequence identity to the DMOT9 (SEQ ID NO 64) sequence. The amino acid sequence of the DMOT9 enzyme may be at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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, suitably at least 90%, more suitably at least 95% to SEQ ID NO 64. In terms of sequence identity, the enzymes defined herein typically retain the function of transferring an acyl unit to the C-4 position of the D-fucose of F*.

[0106] The percentage sequence identity discussed in this application is the percentage sequence identity of the full-length sequence identified by the SEQ.ID NO. This can include a shortened sequence with the same sequence identity measured over the length of the shortened sequence. The shortened sequence can have the same homology as the percentage sequence identity of the SEQ ID NO, regardless of the length of the shortened sequence. The shortened sequence can be at least half the length of the full-length sequence, preferably at least three quarters the length of the full-length sequence.

[0107] In the method of the present invention, the transfer of the rhamnose residue to the C-3 position of the D-fucose of F* can be performed by the enzyme QS-7-RhaT (SEQ ID NO 58) or an enzyme having at least 70% sequence identity with the sequence of QS-7-RhaT. The enzyme is capable of transferring the rhamnose moiety to the C-3 position of the D-fucose of the F* chain. The function of the enzyme can be determined, for example, according to the method described in Example 3.

[0108] The functionality of QS-7-RhaT can be determined by expressing the enzymes required to produce QA-Tri(X / R)-F*-Ac and the QS-7-RhaT candidate in a heterologous host such as N. benthamiana or yeast. The presence of the expected product can be assessed by LC-MS analysis, ultimately supplemented by NMR analysis. Alternatively, in vitro testing may be preferred, where QA-Tri(X / R)-F* is purified from a plant extract or is produced in vitro in an assay containing sapote and the glycosyltransferases necessary to produce QA-Tri(X / R)-F*, or β-amyrin and the enzymes necessary to produce QA-Tri(X / R)-F*. The activity of the candidate QS-7-RhaT is then tested in vitro on the QA-Tri(X / R)-F* substrate, and product formation is determined by LC-MS analysis.

[0109] In the method of the present invention, the transfer of the rhamnose residue to the C-3 position of the D-fucose of F* can be performed 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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 58. Thus, in some embodiments, QS-7-RhaT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 58, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of transferring a rhamnose moiety to the C-3 position of the D-fucose of F*.

[0110] In the method of the present invention, the transfer of a glucose residue to a molecule comprising QA-Tri(X / R)-F*-R-Ac to form QA-Tri(X / R)-F*-GR-Ac is carried out by the enzyme QS-7-GlcT (SEQ ID NO 56) or an enzyme having at least 70% sequence identity with SEQ ID NO 56. The enzyme is capable of transferring a glucose residue to the C-3 position of the rhamnose residue of F*. The function of the enzyme can be determined, for example, according to the method described in Example 1.

[0111] The functionality of QS-7-GlcT can be determined by expressing the enzymes required to produce QA-Tri(X / R)-F*-R-Ac and the QS-7-GlcT candidate in a heterologous host such as N. benthamiana or yeast. The presence of the expected product can be assessed by LC-MS analysis, ultimately supplemented by NMR analysis. Alternatively, in vitro testing may be preferred, where QA-Tri(X / R)-F*-R-Ac is purified from a plant extract or is produced in vitro in an assay containing sapote and the glycosyltransferases necessary to produce QA-Tri(X / R)-F*-R-Ac, or β-amyrin and the enzymes necessary to produce QA-Tri(X / R)-F*-R-Ac. The activity of the candidate QS-7-GlcT is then tested in vitro on the QA-Tri(X / R)-F*-R-Ac substrate, and product formation is determined by LC-MS analysis.

[0112] In the methods of the present invention, the transfer of a glucose residue to a molecule comprising QA-Tri(X / R)-F*-R-Ac to form QA-Tri(X / R)-F*-GR-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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 56. Thus, in some embodiments, QS-7-GlcT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 56, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of transferring a glucose residue to a molecule comprising QA-Tri(X / R)-F*-R-Ac to form QA-Tri(X / R)-F*-GR-Ac.

[0113] The percent 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.

[0114] As described above, the method of the present invention comprises adding an acyl moiety, a glucose moiety and a rhamnose moiety to QA-Tri(X / R)-F*. QA-Tri(X / R)-F* is as described above. An additional feature of the method of the present invention is a step of making a QA backbone, a branched trisaccharide at the C-3 position of the molecule comprising the QA backbone (QA-Tri(X / R)), and a linear sugar chain (F*) at the C-28 position of the molecule comprising the QA backbone (QA-Tri(X / R)-F*).

[0115] QA synthesis

[0116] One step of the method of forming the QA backbone of a molecule comprising QA-Tri(X / R)-F* is cyclizing 2,3-oxidized squalene to form a molecule comprising the triterpene β-amyrin. This step is performed by an oxidized squalene cyclase. In particular, the oxidized squalene cyclase may be an enzyme according to QsbAS (SEQ ID NO 18) or a sequence having at least 50% sequence identity with SEQ ID NO 18. The oxidized squalene cyclase may be encoded by the polynucleotide sequence of SEQ ID NO 17.

[0117] This step encompasses an oxidosqualene cyclase having at least 50% sequence identity to the sequence of QsbAS (SEQ ID NO 18). The amino acid sequence of the QsbAS enzyme may be at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 18. Thus, in some embodiments, QsbAS is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 18, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of cyclizing 2,3-oxidosqualene to form a molecule comprising the triterpene β-amyrin.

[0118] The molecule comprising the β-amyrin scaffold is further oxidized to carboxylic acid, alcohol and aldehyde at C-28, C-16α and C-23, respectively. Another step of this feature of the invention is to oxidize the molecule comprising the β-amyrin scaffold to form a carboxylic acid at C-28. This step is performed by a cytochrome P450 monooxygenase. The cytochrome P450 monooxygenase is a C-28 oxidase QsCYP716-C-28. For example, the C-28 oxidase QsCYP716-C-28 can be according to SEQ ID NO 20 or a sequence having at least 50% sequence identity with SEQ ID NO 20. QsCYP716-C-28 can be encoded by a polynucleotide sequence of SEQ ID NO 19 or a sequence having at least 50% sequence identity with SEQ ID NO 19.

[0119] This step encompasses cytochrome P450 monooxygenases 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 may be at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 20. Thus, in some embodiments, QsCYP716-C-28 is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 20, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the functionality to oxidize molecules comprising the [beta]-amyrin scaffold to form a carboxylic acid at position C-28.

[0120] Another step of this feature of the invention is to oxidize the molecule containing the β-amyrin scaffold to form an alcohol at the C-16 position. This step is performed by a cytochrome P450 monooxygenase. The cytochrome P450 monooxygenase is a C-16α oxidase QsCYP716-C-16α. For example, the C-16α oxidase QsCYP716-C-16α can be according to SEQ ID NO 22 or a sequence having at least 50% sequence identity with 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 with SEQ ID NO 21.

[0121] This step encompasses cytochrome P450 monooxygenases having at least 50% sequence identity to the QsCYP716-16α (SEQ ID NO 22) sequence. The amino acid sequence of the QsCYP716-C-16α enzyme may be at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 22. Thus, in some embodiments, QsCYP716-C-16α is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 22, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the functionality to oxidize molecules comprising the [beta]-amyrin scaffold to form an alcohol at the C-16 position.

[0122] Another step of this feature of the invention is to oxidize the molecule containing the β-amyrin scaffold to form an aldehyde at the C-23 position. This step is performed by a cytochrome P450 monooxygenase. The cytochrome P450 monooxygenase is a C-23 oxidase QsCYP714-C-23. For example, the C-23 oxidase QsCYP714-C-23 can be based on SEQ ID NO 24 or a sequence having at least 50% sequence identity with SEQ ID NO 24. QsCYP714-C-23 can be encoded by a polynucleotide sequence of SEQ ID NO 23 or a sequence having at least 50% sequence identity with SEQ ID NO 23.

[0123] This step encompasses cytochrome P450 monooxygenases 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 may be at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 24. Thus, in some embodiments, QsCYP714-C-23 is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 24, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the functionality to oxidize molecules comprising the [beta]-amyrin scaffold to form an aldehyde at position C-23.

[0124] These steps form the QA skeleton.

[0125] This feature of the invention relates to a method of making a molecule comprising a QA backbone, the method involving a plurality of steps. These steps may be performed in a particular order or in any order or simultaneously. Preferably, the molecule is formed by generating a β-amyrin scaffold followed by sequential oxidation at C-28, C-16α and C-23, respectively. The steps of this feature of these aspects of the invention are described with respect to the preferred case described above. However, the steps may be performed in any order.

[0126] Then the sugar units forming the C-3 branched trisaccharide and F* are added. Preferably, a molecule comprising a QA backbone is made, followed by a step of adding the C-3 chain, followed by a step of adding F*. However, these steps may be performed in a particular order or in any order or simultaneously.

[0127] Synthesis of C-3 branched trisaccharide

[0128] The steps for the formation of Tri(X / R) of a molecule comprising QA-Tri(X / R)-F* are described for the case when the branched trisaccharide at the C-3 position of a molecule comprising the QA backbone is initiated by attaching a β-D-pyranoglucopyranose moiety to a molecule comprising QA to form a molecule comprising QA-Mono. However, these steps may occur in any order.

[0129] The first step in forming the C-3 chain is to attach a β-D-pyranoglucuronic acid moiety to a molecule comprising QA to form a molecule comprising QA-Mono. This step can be performed by an enzyme QsCSL1 according to SEQ ID NO 26, or an enzyme QsCslG2 according to SEQ ID NO 28, or a sequence having at least 70% sequence identity with SEQ ID NO 26 or 28. QsCSL1 can be encoded by a polynucleotide sequence of SEQ ID NO 25, or a sequence having at least 70% sequence identity with SEQ ID NO 25. QsCslG2 can be encoded by a polynucleotide sequence of SEQ ID NO 27, or a sequence having at least 70% sequence identity with SEQ ID NO 27.

[0130] This step encompasses 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 may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 26. The amino acid sequence of the QsCslG2 enzyme may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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% identity, suitably at least 90%, more suitably at least 95% identity to SEQ ID NO 26 or 28. In terms of sequence identity, the enzymes defined herein typically retain the function of attaching a β-D-pyranoglucopyranosyl moiety to a molecule comprising QA to form a molecule comprising QA-Mono.

[0131] Another step of the method of forming the C-3 chain is to attach a D-galactopyranose moiety to a β-D-pyranoglucuronic acid moiety on the molecule comprising QA-Mono to form a molecule comprising QA-Di. This step can be performed by the enzyme Qs-3-O-GalT according to SEQ ID NO 30 or a sequence having at least 70% sequence identity with SEQ ID NO 30. Qs-3-O-GalT can be encoded by a polynucleotide sequence of SEQ ID NO 29 or a sequence having at least 70% sequence identity with SEQ ID NO 29.

[0132] 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 may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 30. Thus, in some embodiments, Qs-3-O-GalT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 30, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein typically retain the function of attaching a D-galactopyranose moiety to a β-D-pyranoglucuronic acid moiety on a molecule comprising QA-Mono to form a molecule comprising QA-Di.

[0133] Another step of the method of forming the C-3 chain is to attach an L-rhamnose moiety to a β-D-pyranoglucuronic acid moiety on a molecule comprising QA-Di to form a molecule comprising QA-TriR. This step can be performed by the enzyme DN20529_c0_g2_i8 according to SEQ ID NO 36, Qs_0283850 according to SEQ ID NO 34, or the enzyme Qs-3-O-RhaT / XylT according to SEQ ID NO 32, or a sequence having at least 70% sequence identity with 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 with 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 with SEQ ID NO 33. Qs-3-O-RhaT / XylT may 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.

[0134] This step encompasses enzymes 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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 36. The amino acid sequence of the Qs_0283850 enzyme may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 34. The amino acid sequence of the Qs-3-O-RhaT / XylT enzyme may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 32. Thus, in some embodiments, DN20529_c0_g2_i8, Qs_0283850 and / or QQs-3-O-RhaT / XylT are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 36, 34 or 32, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein typically retain the function of attaching an L-rhamnose pyranose moiety to a β-D-glucopyranose uronic acid moiety on a molecule comprising QA-Di to form a molecule comprising QA-TriR.

[0135] Yet another step of the method of forming the C-3 chain is to attach a β-D-xylopyranose moiety to a β-D-pyranoglucopyranose moiety on a molecule comprising QA-Di to form a molecule comprising QA-TriX. This step can be performed by an enzyme Qs_0283870 according to SEQ ID NO 38, or an enzyme Qs-3-O-RhaT / XylT according to 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 a 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 a polynucleotide sequence of SEQ ID NO 31, or a sequence having at least 70% sequence identity to SEQ ID NO 31.

[0136] This step encompasses enzymes having at least 70% sequence identity to the sequence of Qs_0283870 or Qs-3-O-RhaT / XylT (SEQ ID NO 38 or 32, respectively). The amino acid sequence of the Qs_0283870 enzyme may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 38. The amino acid sequence of the Qs-3-O-RhaT / XylT enzyme may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 32. Thus, in some embodiments, Qs_0283870 and / or Qs-3-O-RhaT / XylT has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, suitably at least 90%, more suitably at least 95% identity to SEQ ID NO 38 or 32. In terms of sequence identity, the enzymes defined herein typically retain the function of attaching a β-D-xylopyranose moiety to a β-D-pyranoglucuronic acid moiety on a molecule comprising QA-Di to form a molecule comprising QA-TriX.

[0137] These steps form the C-3 chain on the QA backbone.

[0138] Synthesis of C-28 Straight-chain Tetrasaccharide

[0139] The steps for the formation of F* of a molecule comprising QA-Tri(X / R)-F* are described for the case where F* of a molecule comprising a QA backbone is initiated by attaching a UDP-α-D-fucose moiety to a molecule comprising QA-Tri(X / R) to form a molecule comprising QA-Tri(X / R)-F. However, these steps may occur in any order. For example, F* may be generated and then attached to the QA-Tri(X / R) backbone.

[0140] The first step in forming F* can be to attach a UDP-α-D-fucose moiety to the C-28 position of a molecule comprising QA-Tri(R / X) to form a molecule comprising QA-Tri(R / X)-F. This step can be performed by the enzyme Qs-28-O-FucT according to SEQ ID NO 2, or a sequence having at least 70% sequence identity to SEQ ID NO 2. Qs-28-O-FucT can be encoded by a polynucleotide sequence of SEQ ID NO 1, or a sequence having at least 70% sequence identity to SEQ ID NO 1. The first step in forming F* can also be to attach UDP-4-keto, 6-deoxy-D-glucose to a molecule comprising QA-Tri(R / X) to form a molecule comprising QA-Tri(R / X)-F. This step may be performed by the enzyme Qs-28-O-FucT according to SEQ ID NO 2 or a sequence having at least 70% sequence identity with SEQ ID NO 2 and the enzyme QsFucSyn according to SEQ ID NO 12 or a sequence having at least 45% sequence identity with SEQ ID NO 12. Qs-28-O-FucT may be encoded by the polynucleotide sequence of SEQ ID NO 1 or a sequence having at least 70% sequence identity with SEQ ID NO 1. QsFucSyn may be encoded by the polynucleotide sequence of SEQ ID NO 11 or a sequence having at least 45% sequence identity with SEQ ID NO 11.

[0141] This step encompasses enzymes 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 may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 2. Thus, in some embodiments, Qs-28-O-FucT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 2, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of attaching a UDP-α-D-fucose moiety to the C-28 position of a molecule comprising QA-Tri(R / X) to form a molecule comprising QA-Tri(R / X)-F; or the function of attaching UDP-4-keto, 6-deoxy-D-glucose to a molecule comprising QA-Tri(R / X) to form a molecule comprising QA-Tri(R / X)-F.

[0142] This step also encompasses enzymes having at least 45% sequence identity to the sequence of QsFucSyn (SEQ ID NO 12). The amino acid sequence of the QsFucSyn enzyme may be at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 12. Thus, in some embodiments, QsFucSyn is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 12, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of attaching a UDP-α-D-fucose moiety to the C-28 position of a molecule comprising QA-Tri(R / X) to form a molecule comprising QA-Tri(R / X)-F; or the function of attaching UDP-4-keto, 6-deoxy-D-glucose to a molecule comprising QA-Tri(R / X) to form a molecule comprising QA-Tri(R / X)-F.

[0143] Another step in forming F* is to attach a UDP-β-L-rhamnose moiety to a UDP-α-D-fucose moiety on the molecule comprising QA-Tri(R / X)-F to form a molecule comprising QA-Tri(R / X)-FR. This step can be performed by the enzyme Qs-28-O-RhaT according to SEQ ID NO 4, or a sequence having at least 70% sequence identity with SEQ ID NO 4. Qs-28-O-RhaT can be encoded by a polynucleotide sequence of SEQ ID NO 3, or a sequence having at least 70% sequence identity with SEQ ID NO 3.

[0144] This step also encompasses 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 may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 4. Thus, in some embodiments, Qs-28-O-RhaT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 4, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of attaching a UDP-β-L-rhamnose moiety to a UDP-α-D-fucose moiety on a molecule comprising QA-Tri(R / X)-F to form a molecule comprising QA-Tri(R / X)-FR.

[0145] A further step of forming F* is to attach a UDP-α-D-xylose moiety to a UDP-β-L-rhamnose moiety on a molecule comprising QA-Tri(R / X)-FR to form a molecule comprising QA-Tri(R / X)-FRX. This step can be performed by the enzyme Qs-28-O-XylT3 according to SEQ ID NO 2 or a sequence having at least 70% sequence identity with SEQ ID NO 6. Qs-28-O-XylT3 can be encoded by a polynucleotide sequence of SEQ ID NO 5 or a sequence having at least 70% sequence identity with SEQ ID NO 5.

[0146] This step also encompasses enzymes 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 may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 6. Thus, in some embodiments, Qs-28-O-XylT3 is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 6, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of attaching a UDP-α-D-xylose moiety to a UDP-β-L-rhamnose moiety on a molecule comprising QA-Tri(R / X)-FR to form a molecule comprising QA-Tri(R / X)-FRX.

[0147] An optional step of forming F* may be to attach a UDP-α-D-xylose moiety to a UDP-α-D-xylose moiety on a molecule comprising QA-Tri(R / X)-FRX to form a molecule comprising QA-Tri(R / X)-FRXX. This step may be performed by the enzyme Qs-28-O-XylT4 according to SEQ ID NO 8, or a sequence having at least 70% sequence identity to SEQ ID NO 8. Qs-28-O-XylT4 may be encoded by a polynucleotide sequence of SEQ ID NO 7, or a sequence having at least 70% sequence identity to SEQ ID NO 7.

[0148] This optional step also encompasses enzymes 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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 8. Thus, in some embodiments, Qs-28-O-XylT4 is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 8, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of attaching a UDP-α-D-xylose moiety to a UDP-α-D-xylose moiety on a molecule comprising QA-Tri(R / X)-FRX to form a molecule comprising QA-Tri(R / X)-FRXX.

[0149] Another optional step to form F* can be to attach a UDP-α-D-apiose moiety to a UDP-α-D-xylose moiety on a molecule comprising QA-Tri(R / X)-FRX to form a molecule comprising QA-Tri(R / X)-FRXA. This step can be performed by the enzyme Qs-28-O-ApiT4 according to 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 a polynucleotide sequence of SEQ ID NO 9, or a sequence having at least 70% sequence identity to SEQ ID NO 9.

[0150] This step also encompasses enzymes 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 may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 10. Thus, in some embodiments, Qs-28-O-ApiT4 is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 10, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of attaching a UDP-α-D-apiose moiety to a UDP-α-D-xylose moiety on a molecule comprising QA-Tri(R / X)-FRX to form a molecule comprising QA-Tri(R / X)-FRXA.

[0151] These steps form F* on the QA backbone.

[0152] The method of the second aspect of the present invention is carried out in a biological system or host. Polynucleotides encoding one or more of the above enzymes are introduced and expressed in the biological system. In most cases, the biological system will not naturally express any enzyme of the second aspect of the present invention, so the biological system will be engineered to express all of these enzymes.

[0153] The biological system can be a plant or a microorganism. When the biological system is a plant, the plant can be a row crop, such as sunflower, potato, rape (canola), dry beans, peas, flax, safflower, buckwheat, cotton, cereals, soybeans and sugar beets. The plant can also be corn, wheat, rape and rice. Preferably, the plant can be Nicotiana benthamiana.

[0154] In certain embodiments of the methods of the second aspect of the invention, the biological system is not Quillaja saponaria.

[0155] When the biological system is a microorganism, the microorganism may be a bacterium or a yeast.

[0156] Yeast (Saccharomyces cerevisiae) is a heterologous host for the production of high-value small molecules, including terpenes. Similar to plants, yeast endogenously produces the triterpenoid precursor 2,3-squalene oxide and is therefore a promising host for the industrial-scale production of triterpenoids. It is also an efficient host for the functional expression of plant CYPs on the endoplasmic reticulum membrane. Endogenous yeast enzymes cause minimal modification of the triterpene scaffold, thereby facilitating product purification. Yeast can serve as a production host for the production of triterpenes with a variety of glycosidic conjugates containing a variety of sugar types in both linear and branched configurations. Glycosylation reactions in yeast are limited by the limited palette of endogenous sugar donors. However, by expressing genes from higher plants, the nucleotide sugar metabolism of yeast can be expanded beyond UDP-glucose and UDP-galactose to include UDP-rhamnose, UDP-glucuronic acid, UDP-xylose, UDP-arabinose, etc.

[0157] The method of the first aspect of the invention can be performed in vitro. In the sense of the present invention, "in vitro" refers to enzymatic treatment of an appropriate QA-Tri(X / R)-F* derivative with an appropriate enzyme of the invention. The QA-Tri(X / R)-F* derivative can be produced by biosynthesis or by chemical synthesis. The enzyme can be cloned or purified from its native environment. It is within the capabilities of those skilled in the art to determine the optimal conditions for the enzymatic treatment (e.g., duration, temperature, buffer, etc.).

[0158] The identity of the QA derivative can be confirmed, for example, by elucidating its structure by NMR, as described in Materials and Methods.

[0159] In the second, third and fourth aspects of the invention, the amino acid sequence SEQ ID NO 60 is encoded by the polynucleotide sequence SEQ ID NO 59; the amino acid sequence SEQ ID NO 58 is encoded by the polynucleotide sequence SEQ ID NO 57; and the amino acid sequence SEQ ID NO 56 is encoded by the polynucleotide sequence SEQ ID NO 55.

[0160] The methods of the second, third and fourth aspects of the invention comprise introducing a polynucleotide required for the biosynthesis of a molecule comprising QA-Tri(X / R)-F*-GR-Ac into a host cell via a vector, thereby transforming the host with the polynucleotide. Recombination may occur between the vector and the host cell genome to introduce the polynucleotide into the host cell genome.

[0161] The fifth aspect of the present invention is a glucosyltransferase (QS-7-GlcT) according to SEQ ID NO 56 or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO 56. The enzyme is capable of transferring a glucose residue to the C-3 position of the rhamnose residue of F* of the QS-7 precursor. The enzyme is as described in the method of the first to fourth aspects of the present invention and has the same properties and functions as described in the method of the first to fourth aspects of the present invention.

[0162] The glucosyltransferase may be encoded by the polynucleotide of SEQ ID NO 55 or a polynucleotide molecule that also encodes an amino acid according to the fifth aspect of the invention. The QS-7-GlcT enzyme may, for example, be encoded by a polynucleotide sequence according to SEQ ID NO 55 or by a sequence that also encodes an enzyme according to the fifth aspect of the invention according to degenerate codons.

[0163] The fifth aspect of the invention encompasses a glucosyltransferase 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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 56. Thus, in some embodiments, QS-7-GlcT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 56, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein generally retain the function of transferring a glucose moiety to a molecule comprising QA-Tri(X / R)-F* to form QA-Tri(X / R)-F*G.

[0164] The sixth aspect of the present invention is a rhamnosyltransferase (QS-7-RhaT) according to SEQ ID NO 58 or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 58. The enzyme is capable of transferring a rhamnose moiety to the C-3 position of the D-fucose of F* of the QS-7 precursor. The enzyme is as described in the method of the first to fourth aspects of the present invention and has the same properties and functions as described in the method of the first to fourth aspects of the present invention.

[0165] The rhamnosyltransferase may be encoded by the polynucleotide of SEQ ID NO 57 or a polynucleotide molecule that also encodes an amino acid according to the sixth aspect of the invention. The QS-7-RhaT enzyme may, for example, be encoded by a polynucleotide sequence according to SEQ ID NO 57 or by a sequence that also encodes an enzyme according to the sixth aspect of the invention according to degenerate codons.

[0166] A sixth aspect of the invention encompasses a rhamnosyltransferase 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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 58. Thus, in some embodiments, QS-7-RhaT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 58, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein typically retain the function of transferring a rhamnose moiety to the C-3 position of the D-fucose of F* of the QS-7 precursor.

[0167] The seventh aspect of the present invention is an acetyltransferase (QS-7-AcetylT) according to SEQ ID NO 60 or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO 60. The enzyme is capable of transferring an acyl unit to the C-4 position of the D-fucose of F* of the QS-7 precursor. The enzyme is as described in the method of the first to fourth aspects of the present invention and has the same properties and functions as described in the method of the first to fourth aspects of the present invention.

[0168] The acetyltransferase may be encoded by the polynucleotide of SEQ ID NO 59 or a polynucleotide molecule that also encodes an amino acid according to the seventh aspect of the invention. The QS-7-AcetylT enzyme may, for example, be encoded by a polynucleotide sequence according to SEQ ID NO 59 or by a sequence that also encodes an enzyme according to the seventh aspect of the invention according to degenerate codons.

[0169] The seventh aspect of the invention encompasses an acetyltransferase 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 be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 60. Thus, in some embodiments, QS-7-AcetylT is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 60, suitably at least 90%, more suitably at least 95%. In terms of sequence identity, the enzymes defined herein typically retain the function of transferring an acyl unit to the C-4 position of the D-fucose of F* of the QS-7 precursor.

[0170] Any percent sequence identity of the fifth, sixth and seventh aspects of the invention may be combined with any other percent sequence identity of the fifth, sixth and seventh aspects of the invention.

[0171] The eighth aspect of the present invention is a polynucleotide encoding one or more enzymes of the fifth to seventh aspects of the present invention.

[0172] The ninth aspect of the present invention is a vector comprising one or more polynucleotides according to the eighth aspect of the present invention.

[0173] The vector may comprise one, two or three polynucleotides encoding the enzymes of the fifth to seventh aspects of the invention. Preferably, the vector will comprise three polynucleotides encoding the enzymes of the fifth to seventh aspects of the invention, or a plurality of vectors comprising these three polynucleotides together.

[0174] The tenth aspect of the present invention is a host cell comprising one or more polynucleotides according to the eighth aspect of the present invention.

[0175] The host cell can 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, the plant is preferably Nicotiana benthamiana.

[0176] The additional 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 can be introduced into the host cell through a vector. Recombination can occur between the vector and the host cell genome to introduce the polynucleotide into the host cell genome. Alternatively, the polynucleotide can be introduced into the host cell by co-infiltration with multiple recombinant vectors. The recombinant vector can be an Agrobacterium tumefaciens strain, as described below.

[0177] The eleventh aspect of the present invention is a host cell transformed with the vector described in the ninth aspect of the present invention.

[0178] The twelfth aspect of the present invention is a plant or microbial biosystem comprising a host cell as described in the tenth and eleventh aspects of the present invention. The biosystem can be a plant or a microorganism. When the biosystem is a plant, it can be Nicotiana benthamiana or any of the above plants. The method for producing the plant comprises the steps of introducing the polynucleotide of the present invention into a host plant cell and regenerating the plant from the transformed host plant cell. When the biosystem is a microorganism, it can be a yeast.

[0179] The present invention also includes methods for making each enzyme and each polynucleotide of the above aspects of the present invention, as well as methods for making a vector comprising one or more polynucleotides of the present invention, and methods for making the host cell of the tenth and eleventh aspects of the present invention and the biological system of the twelfth aspect of the present invention. These methods use techniques and products well known in the art, such as those in WO2019 / 122259 and WO2020 / 260475, and are described in more detail as follows:

[0180] The polynucleotides of the present invention may be included in a vector, particularly an expression vector. The vector may be any plasmid, cosmid, phage or Agrobacterium vector that is double-stranded or single-stranded linear or circular, which may be integrated into the cell genome or otherwise transform a prokaryotic or eukaryotic host. The vector may be an expression vector, including an inducible promoter operably linked to a polynucleotide sequence. Typically, the vector may include an enhancer sequence between the inducible promoter and the polynucleotide sequence. The vector may also include a terminator sequence and an optional 3'UTR upstream of the terminator sequence. The vector may include one or more polynucleotides encoding the enzyme of the fifth to seventh aspects of the present invention, preferably including all sequences required for producing a version of the molecule described in the first and second aspects of the present invention. The vector may be a plant vector or a microbial vector.

[0181] The polynucleotide in the vector can be controlled by or operably connected to an appropriate promoter or other regulatory elements to transcribe in a host cell. The host cell can be a yeast cell, a bacterial cell or a plant cell. The vector can be a bifunctional expression vector that plays a role in a variety of hosts. For genomic DNA, this can include its own promoter or other regulatory elements. The advantage of using a natural promoter is that pleiotropic reactions can be avoided. For cDNA, this can be controlled by an appropriate promoter or other regulatory elements to express in a host cell.

[0182] Preferred vectors for use in plants contain border sequences that allow transfer and integration of the expression vector into the plant genome. The vector may be a plant binary vector.

[0183] The vector can be transfected into a host cell in any biological system. The host can be a microorganism, such as Escherichia coli or yeast. The vector can be part of an Agrobacterium tumefaciens strain and used to infect a biological plant host system. Agrobacterium tumefaciens can each contain one of the polynucleotides encoding the present invention, and can be combined to co-infect the host cell so that the host cell contains all the polynucleotides required for the enzyme encoding the fifth to seventh aspects of the present invention.

[0184] The present invention also includes the steps of culturing or growing the host to produce, harvest and isolate the desired QA-Tri(X / R)-F*-GR-Ac derivative.

[0185] An additional feature of the first to fourth aspects of the present invention is the step of isolating the QA-Tri(X / R)-F*-GR-Ac derivative.

[0186] The thirteenth aspect of the present invention is a QA-Tri(X / R)-F*-GR-Ac derivative obtained by the method of the present invention, in particular the method of the first to fourth aspects of the present invention. The QA-Tri(X / R)-F*-GR-Ac derivative obtained by the method of the present invention can 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 of this aspect of the present invention can be obtained by the method of the present invention. The QA-Tri(X / R)-F*-GR-Ac derivative may preferably be QA-TriX-FRXA-GR-Ac.

[0187] Another aspect of the invention is a method for making a QA-Tri(X / R)-F*-GR-Ac derivative comprising the method steps of the invention including the step of isolating the QA derivative.

[0188] A fourteenth aspect of the present invention is the use of QA-Tri(X / R)-F*-GR-Ac derivatives, in particular QA-TriX-FRXA-GR-Ac, as adjuvants to be included in vaccine compositions after isolation from biological systems. The adjuvant may be a liposome formulation or an immunostimulatory complex (ISCOM) formulation.

[0189] An additional feature of the fourteenth aspect of the present invention is that the adjuvant also comprises a TLR4 agonist. The TLR4 agonist can be 3D-MPL. The QA-Tri (X / R) -F * -GR-Ac derivative of the present invention can be combined with other immunostimulants, such as TLR4 agonists, in particular lipopolysaccharide TLR4 agonists, such as lipid A derivatives, especially monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL). 3D-MPL is sold by GlaxoSmithKline Biologicals NA under the name of "MPL". For example, see 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 GB 2220 211A. Chemically, it is a mixture of 3-deacetylated monophosphoryl lipid A with 4, 5 or 6 acetylated chains.

[0190] Other TLR4 agonists that can be combined with the QA derivatives of the invention include glucose-based lipid adjuvants (GLA), such as described in WO2008 / 153541 or WO2009 / 143457 or in the literature (Coler et al. 2011 and Arias et al. 2012).

[0191] An additional feature of the fourteenth aspect of the invention is the combination of a QA-Tri(X / R)-F*GR-Ac derivative (e.g., QA-Tri(X / R)-FRXA-GR-Ac) with QS-21 (either as a purified fraction from the bark of Quillaja saponaria or produced biosynthetically).

[0192] The adjuvants of the invention may also be formulated into suitable carriers, such as emulsions (eg, oil-in-water emulsions), liposomes, or immunostimulatory complexes (ISCOMs), as described below.

[0193] Liposomes

[0194] The term "liposome" is well known in the art and defines a general class of vesicles comprising one or more lipid bilayers surrounding an aqueous space. Thus, liposomes are composed of one or more lipid and / or phospholipid bilayers and may contain other molecules, such as proteins or carbohydrates, in their structure. Due to the presence of a lipid phase and an aqueous phase, liposomes may encapsulate or encapsulate water-soluble materials, fat-soluble materials and / or amphiphilic compounds. A method for making such liposomes is described in WO2013 / 041572.

[0195] Liposomes can vary in size from 30 nanometers to several micrometers, depending on the phospholipid composition and method used for their preparation.

[0196] The liposome size will be in the range of 50 nm to 200 nm, particularly 60 nm to 180 nm, for example 70 to 165 nm. Optimally, the liposomes should be stable and have a diameter of 100 nm to allow convenient sterilization by filtration.

[0197] The structural integrity of the liposomes can be assessed by methods such as dynamic light scattering (DLS) measuring the size (Z average diameter, Zav) and polydispersity of the liposomes, or by analyzing the structure of the liposomes by electron microscopy. The average particle size may be between 95 and 120 nm, and / or the polydispersity (Pdl) index may not exceed 0.3 (e.g., not exceed 0.2).

[0198] ISCOM

[0199] The term immunostimulatory complex (ISCOM) is well known in the art and defines a system for delivering an antigen and an adjuvant together in the same particle. ISCOMs are spherical, hollow, cage-like, self-assembled particles.

[0200] Saponin-based adjuvants can be formulated in ISCOM and / or ISCOM-Matrix structures. ISCOM can be prepared according to the methods described in EP0109942B1, WO87 / 02250 and EP0180546B1. Transport and / or passenger antigens can be used, as described in WO9730728A1.

[0201] ISCOM can be an ISCOM matrix complex comprising at least one saponin fraction and a lipid. The lipid can be a sterol, such as cholesterol. The ISCOM matrix complex can also contain a phospholipid, such as phosphatidylcholine. The ISCOM matrix complex can also contain one or more other immunomodulatory (adjuvant activity) substances and can be produced according to the method described in EP0436620B1. The ISCOM matrix can be formulated into a mixture with an antigen, and the association between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0202] ISCOMs may be ISCOM complexes containing at least one saponin, at least one lipid and at least one type of antigen or epitope. ISCOM complexes contain antigens associated by detergent treatment such that a portion of the antigen is incorporated into the particle.

[0203] In some embodiments, the saponin fraction or at least one additional adjuvant is selected from a QA derivative QA-Tri(X / R)-F*-GR-Ac (e.g., QA-Tri(X / R)-FRXA-GR-Ac) or QS-21 (a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or any purified subfraction).

[0204] Each ISCOM particle may contain one or at least two saponin fractions. The ISCOM particle may contain at least two saponin fractions of the same or different weight %. For example, the particle may contain any weight % of the QA derivative QA-Tri (X / R) -F * -GR-Ac and any weight % of another saponin fraction, such as QS-21. Thus, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1 to 99.9 weight %, 5 to 95 weight %, 10 to 90 weight %, 15 to 85 weight %, 20 to 80 weight %, 25 to 75 weight %, 30 to 70 weight %, 35 to 65 weight %, 40 to 60 weight %, 45 to 55 weight %, 40 to 60 weight % or 50 weight % of a saponin fraction, such as the QA derivative QA-Tri (X / R) -F * -GR-Ac and in each case the balance is made up to 100% of another saponin, such as QS-21. This weight is calculated based on 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.

[0205] The ISCOM matrix or ISCOM complex may contain 5-99% by weight of one fraction (e.g., QA derivative QA-Tri (X / R)-F*-GR-Ac) and the balance of another fraction (e.g., QS-21) to make up to 100% by weight. The ISCOM matrix or ISCOM complex may contain at least two saponin fractions of the same or different weight percentages. The weight is calculated based on the total weight of the saponin fractions. The ISCOM matrix or ISCOM complex may contain 40% to 99% by weight of one fraction (e.g., QA derivative QA-Tri (X / R)-F*-GR-Ac) and 1% to 60% by weight of another fraction (e.g., QS-21). The ISCOM matrix or ISCOM complex may contain 70% to 95% by weight of one fraction (e.g., QA derivative QA-Tri (X / R)-F*-GR-Ac) and 30% to 5% by weight of another fraction (e.g., QS-21).

[0206] ISCOM matrix particles and ISCOM composite particles can each be formed using only one saponin fraction. The composition can contain multiple particles and each particle can contain only one saponin fraction. The composition can contain one or more different types of particles (e.g., ISCOM-matrix complex particles, ISCOM complex particles), wherein each individual particle contains one saponin fraction. The saponin fraction in one particle can be different from the saponin fraction in other particles.

[0207] One type of saponin fraction or crude saponin fraction may be incorporated into one ISCOM matrix complex or particle, while another type of saponin fraction or crude saponin fraction may be incorporated into another ISCOM matrix complex or particle. The composition or vaccine may comprise at least two types of complexes or particles, each type incorporating one type of saponin into physically distinct particles.

[0208] In the composition, ISCOM matrix composite particles and / or mixtures of ISCOM composite particles may be used, wherein the two saponin fractions are separately incorporated into different ISCOM matrix composite particles and / or ISCOM composite particles.

[0209] A composition may contain ISCOM matrix or ISCOM composite particles, each containing a saponin fraction. The composition may contain particles of different or equal weight %. For example, the composition may contain 0.1% to 99.9% by weight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight, 20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to 65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40% to 60% by weight, or 50% by weight of an ISCOM matrix or composite containing a first saponin fraction, with the remainder consisting of an ISCOM matrix or composite containing a different saponin fraction.

[0210] The saponin fraction in the first ISCOM matrix or ISCOM composite particle may be the QA derivative QA-Tri(X / R)-F*-GR-Ac and the saponin fraction in the second ISCOM matrix or ISCOM composite particle may be QS-21.

[0211] A preferred composition comprises a first ISCOM matrix comprising a QA derivative QA-Tri(X / R)-F*-GR-Ac, and a second ISCOM matrix comprising QS-21, wherein the first ISCOM matrix comprises about 70% by weight of the total saponin adjuvant and the second ISCOM matrix comprises about 30% by weight of the total saponin adjuvant. Another preferred composition comprises a first ISCOM matrix comprising a QA derivative QA-Tri(X / R)-F*-GR-Ac, and a second ISCOM matrix comprising QS-21, wherein the first ISCOM matrix comprises about 85% by weight of the total saponin adjuvant and the second ISCOM matrix comprises about 15% by weight of the total saponin adjuvant. Thus, in certain compositions, the first ISCOM matrix is ​​present in a range of about 70% to about 85% of the total weight of the saponin adjuvant in the composition, and the second ISCOM matrix is ​​present in a range of about 15% to about 30% of the total weight of the saponin adjuvant in the composition.

[0212] The saponin-based adjuvant may be Matrix-M TMAdjuvant. Matrix-M TM The adjuvant can be extracted from the Molina soap bark tree. The adjuvant can be formulated and purified with cholesterol and phospholipids. TM It may consist of two groups of separately formed particles, which may have complementary properties. The particles may be about 25-55 nm, about 30-50 nm or about 35-45 nm, preferably the particles are 40 nm.

[0213] Matrix-M TM One particle can be the QA derivative QA-Tri(X / R)-F*-GR-Ac (particle 1), and the other particle can be QS-21 (particle 2). TM Two particles may be included in the ratio required to maintain high adjuvant activity and an optimal safety margin. For example, Matrix-M TM Contains 85% of Particle 1 and 15% of Particle 2. Matrix-M TM Contains 92% of Particle 1 and 8% of Particle 2.

[0214] Matrix-M TM The adjuvant can be administered at a dose of about 1 to about 100 μg, about 5 to about 95 μg, about 10 to about 90 μg, about 15 to about 85 μg, about 20 to about 80 μg, about 25 to about 75 μg, about 30 to about 70 μg, about 35 to about 65 μg, about 40 to about 60 μg, about 45 to about 55 μg, about 50 μg, or any value therebetween.

[0215] Matrix-M TM The adjuvant induces high and sustained levels of broadly reactive antibodies supported by balanced TH1 and TH2 type responses, including biologically active antibody isotypes such as murine IgG2a, multifunctional T cells and cytotoxic T lymphocytes. TM Adjuvants can enhance the immune response and promote rapid and profound effects on cell drainage to local lymph nodes, thereby creating an environment for activated cells (including T cells, B cells, natural killer cells, neutrophils, monocytes and dendritic cells). TM Can enhance the binding of antibodies and cellular immune responses.

[0216] The fifteenth aspect of the present invention is an adjuvant composition comprising the QA-Tri(X / R)-F*-GR-Ac derivative or QA-TriX-FRXA-GR-Ac of the thirteenth aspect of the present invention.

[0217] Example

[0218] The invention is further described with reference to the following non-limiting examples.

[0219] Example 1 - Identification of QS-7-GlcT

[0220] Previously, a series of genome and transcriptome sequence resources of Quillaja saponaria were generated and used to identify genes required for the production of saponin QA-Tri(X / R)-FRX(A / X) ( Figure 4 ). This saponin is the common precursor to different immunostimulatory saponins produced by Quillaja saponaria, including QS-21 and QS-7. Through these sequence resources, it has been determined that the genes required for the biosynthesis of this scaffold are co-expressed between different Quillaja tissues and are highly expressed in leaf primordia.

[0221] UDP-dependent glycosyltransferases (UGTs) are commonly implicated in the glycosylation of plant natural products, and several such enzymes are known to be required for the production of QA-Tri(X / R)-FRX(A / X). Using the sequence resources described above, one UGT (QsUGT-BI) was identified that exhibited an expression pattern similar to previously characterized enzymes. Transient expression of QsUGT-BI with the Quillaja genes required for the biosynthesis of the QA-TriX-FRXA scaffold (Table 2) resulted in the identification of a new product by LC-MS, the mass of which indicated the addition of a hexose residue ( Figure 5 Several identified saponins from Quillaja saponaria are characterized by having a glucose residue attached to the sugar chain at C-28, suggesting that the hexose added by QsUGT-BI is likely glucose. One such saponin is QS-7, which is characterized by a D-glucose residue attached to the C-3 position of the rhamnose residue at C-28 ( Figure 1 The resulting product was deduced to be QA-TriX-FRXA glucoside (QA-TriX-FRXA-G), which is considered to be a precursor of QS-7. The deduced glucosyltransferase QsUGT-BI is also referred to herein as QS-7-GlcT.

[0222] Example 2 - Identification of QS-7-AcetylT

[0223] QS-7 is characterized by an acetyl group attached to the C-4 position of D-fucose ( Figure 1). BAHD acyltransferases are known to be commonly involved in the acylation of various plant specialized metabolites. Therefore, a series of BAHD acyltransferases (ACTs) showing co-expression with known QA-TriX-FRXA-G pathway genes were cloned and tested in Nicotiana benthamiana by co-infiltrating ACT candidates with genes required for the biosynthesis of the QA-TriX-FRXA scaffold. After LC-MS analysis of leaf extracts, a new product expressing the candidate "QsACT-19'" was detected in the samples. The mass of this product was found to be QA-TriX-FRXA plus an acetyl group (QA-TriX-FRXA-Ac), indicating that QsACT-19' is an acetyltransferase ( Figure 6 ). Therefore, the putative QA-TriX-FRXA-Ac product is considered to be a QS-7 precursor. QsACT-19' is also referred to herein as QS-7-AcetylT.

[0224] Example 3 - Identification of QS-7-RhaT

[0225] Analysis of known saponins from Quillaja saponaria indicated that the presence of the additional rhamnose (attached to the C-3 position of D-fucose) found in QS-7 appears to be dependent on the presence of an acetyl group attached to C-4 of D-fucose. The product of the QsACT-19' enzyme described above (QA-TriX-FRXA-Ac), which features an acetyl group at the same position as QS-7, was used as a scaffold to screen further UGT pools by transient expression. Of the 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 presumed to be QA-TriX-FRXA-Ac rhamnoside (QA-TriX-FRXA-R-Ac). QsUGT-0023500 is also referred to herein as Qs-7-RhaT.

[0226] Example 4 - Production of QS-7 (QA-TriX-FRXA-GR-Ac)

[0227] After the identification of QsUGT-BI, QsUGT-0023500, and QsACT-19', the constructs required for the production of QA-TriX-FRXA were combined with the three newly identified genes and another round of transient expression was performed. Subsequent LC-MS analysis of leaf extracts showed peaks that matched the retention time and mass spectrum of the QS-7 standard, a fraction purified from the bark of the Quillaja sapodilla tree ( Figure 8). This peak was not present in any of the control samples without any of the three newly identified enzymes. This seems to confirm the relevance of these enzymes for QS-7 production. Nevertheless, in order to determine the unequivocal QS-7 production, a large-scale infiltration was performed using a total of 410 plants. Leaf material extraction allowed the isolation of the new product by semi-preparative HPLC. 1 H NMR analysis confirmed the identity of the new product ( Fig. 9 and Fig.10 ).

[0228] Materials and methods

[0229] Primers and cloning

[0230] 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 Quillaja saponaria. PCR was performed using the primers detailed in Table 1 and iProof polymerase with thermal cycling according to the manufacturer's recommendations. The resulting PCR products were purified (Qiagen PCR cleanup kit) and cloned into pDONR207 vectors 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 verify the presence of the correct genes. Next, the three genes were further subcloned into the pEAQ-HT-DEST1 expression vector using LR clonase. The resulting vectors were transformed into A. tumefaciens LBA4404 by liquid nitrogen flash freezing.

[0231]

[0232] Table 1 Primers used to clone the genes described herein. Gene specific sequences are shown in black, while The attB site required for cloning is shown in grey.

[0233] Agrobacterium Infiltration of Nicotiana benthamiana Leaves

[0234] Agrobacterium infiltration was performed using a needleless syringe as previously described (Reed et al., 2017). All genes were expressed from the pEAQ-HT-DEST1 binary expression vector in Agrobacterium tumefaciens LBA4404 as described above. In some cases, multiple genes were integrated into a single Golden Gate binary vector to facilitate infiltration. Bacteria and plants were cultured as described in (Reed et al., 2017).

[0235] Preparation of Nicotiana benthamiana leaf extract for LC-MS analysis

[0236] Leaves were harvested five days after Agrobacterium infiltration and freeze-dried. The dried leaf material (10 mg per sample) was disrupted with tungsten beads at 1000 rpm for 1 minute (Geno / Grinder 2010, Spex SamplePrep). Metabolites were extracted into 550 μL 80% methanol containing 20 μg / mL internal standard (digoxigenin (Sigma-Aldrich)) and incubated at 18°C ​​for 20 minutes with shaking at 1400 rpm (Thermomixer Comfort, Eppendorf). Each sample was separated twice with 400 μL hexane to defat. 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 suspended in 75 μL 100% methanol and filtered at 12,500xg for 30 seconds (0.2 μm, Spin-X, Costar). The filtrate (50 μL) was combined with 50 μL of 50% methanol in a glass vial and analyzed as detailed below.

[0237] HPLC-CAD-MS Analysis of Nicotiana benthamiana Leaf Extract

[0238] Sample analysis was performed using a QExactive mass spectrometer (Thermo Fisher) equipped with a 50x2.1mm 2.6μ Kinetix XB-C18 column (Phenomenex). The solvent system consisted of water [A] and acetonitrile [B], both containing 0.1% formic acid, with a flow rate of 0.6mL / min. The program included 15% [B] for 0.75 minutes, then gradually increased to 60% [B] until 13 minutes. The percentage of [B] increased to 100% in 0.25 minutes and maintained for 1 minute, then returned to 15% in 0.25 seconds and maintained for 2 minutes. The samples were monitored by CAD and MS set to negative mode with a range of 400-2500 m / z.

[0239] Large-scale vacuum penetration of Nicotiana benthamiana

[0240] Plants were vacuum infiltrated using previously described methods (Reed et al., 2017; Stephenson et al., 2018) with Agrobacterium tumefaciens strain LBA4404 carrying the pEAQ-HT-DEST1 expression vector containing the relevant genes detailed in Table 2.

[0241] Purification of QS-7 from large-scale infiltrated Nicotiana benthamiana

[0242] A series of Agrobacterium tumefaciens cultures containing constructs relevant to QS-7 production were co-infiltrated into N. benthamiana by large-scale vacuum infiltration. A total of 410 plants were agroinfiltrated and leaves were harvested and freeze-dried five days later to yield 104 g of dry material. The leaf material was first defatted with hexane and then subjected to exhaustive extraction with methanol. The methanol extracts were combined and evaporated under reduced pressure. The dried extracts were dissolved in a minimum amount of methanol and diluted with an equal volume of water and then separated in a separatory funnel using a series of hexane, dichloromethane, ethyl acetate, and n-butanol. The butanol layer was collected, dried over anhydrous NaSO4, evaporated under reduced pressure, and redissolved in a minimum amount of methanol. QS-7 was purified by reverse phase semi-preparative HPLC using a Luna C18 column 250x10 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 consisted of 25% [B] for the first 2 minutes, followed by a gradient from 25% to 70% over 18 minutes and a hold at 70% for 5 minutes. The column was equilibrated at 25% [B] for 2 minutes between each run. This provided a pure semi-purified fraction (approximately 12 mg of light brown amorphous material) which was purified according to 1 HNMR analysis showed that it contained 3-5% QS-7.

[0243] NMR analysis

[0244] 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, respectively. Chemical shifts are relative to the residual signal solvent (MeOH-d4: δH 3.31; δC 49.15).

[0245]

[0246] Table 2

[0247] Terms

[0248] Embodiments of the invention are set out in the claims and the following clauses.

[0249] 1. A method for producing biosynthetic QA-Tri(X / R)-F*-GR-Ac in a host, the method comprising the steps of:

[0250] a) expressing genes required for the biosynthesis of QA-TriR-F* and / or QA-TriX-F* into the host, and

[0251] b) introducing into the host a polynucleotide encoding:

[0252] i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0253] 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 with SEQ ID NO 60; and

[0254] 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 with SEQ ID NO 58.

[0255] 2. The method according to item 1, wherein Tri(X / R) is TriX and F* is FRXA.

[0256] 3. The method according to clause 1, wherein step a) comprises:

[0257] 1) expressing the genes required for the biosynthesis of QA-TriR and / or QA-TriX into the host, and

[0258] 2) Introducing into the host a polynucleotide encoding:

[0259] i. Quillaja 28-O-fucosyltransferase (Qs-28-O-FucT, SEQ ID NO 2) or an enzyme having a sequence with at least 70% sequence identity to SEQ ID NO 2, optionally an enzyme from Quillaja spp. promoting the production of fucosylated saponins (QsFucSyn, SEQ ID NO 12) or an enzyme having a sequence with at least 45% sequence identity to SEQ ID NO 12;

[0260] ii. saponin 28-O-fucoside [1,2] -rhamnosyltransferase (Qs-28-O-RhaT, SEQ ID NO 4) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 4;

[0261] iii. saponin 28-O-fucoside [1,2] -rhamnoside [1,4] xylosyltransferase (Qs-28-O-XylT3, SEQ ID NO 6) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 6; and

[0262] iv. Optionally, saponin 28-O-fucoside [1,2]-rhamnoside [1,4]xyloside [1,3]xylosyltransferase (Qs-28-O-XylT4, SEQ ID NO 8) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 8 and / or saponin 28-O-fucoside [1,2]-rhamnoside [1,4]xyloside [1,3]apiacetoglycosyltransferase (Qs-28-O-ApiT4, SEQ ID NO 10) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 10.

[0263] 4. The method according to clause 1, wherein step a) comprises:

[0264] 1) expressing the genes required for the biosynthesis of QA-TriR and / or QA-TriX into the host, and

[0265] 2) Introducing into the host a polynucleotide encoding:

[0266] i. Qs-28-O-FucT (SEQ ID NO 2) or an enzyme having a sequence with at least 70% sequence identity, alternatively QsFucSyn (SEQ ID NO 12) or an enzyme having a sequence with at least 45% sequence identity;

[0267] ii. Qs-28-O-RhaT (SEQ ID NO 4) or an enzyme having a sequence with at least 70% sequence identity;

[0268] iii. Qs-28-O-XylT3 (SEQ ID NO 6) or an enzyme having a sequence with at least 70% sequence identity; and

[0269] iv. Optionally, Qs-28-O-ApiT4 (SEQ ID NO 10) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 10.

[0270] 5. The method according to clause 1, wherein step a) comprises:

[0271] 1) expressing the genes required for the biosynthesis of QA-TriR and / or QA-TriX into the host, and

[0272] 2) Introducing into the host a polynucleotide encoding:

[0273] i. Qs-28-O-FucT (SEQ ID NO 2) or an enzyme having a sequence with at least 70% sequence identity, alternatively QsFucSyn (SEQ ID NO 12) or an enzyme having a sequence with at least 45% sequence identity;

[0274] ii. Qs-28-O-RhaT (SEQ ID NO 4) or an enzyme having a sequence with at least 70% sequence identity;

[0275] iii. Qs-28-O-XylT3 (SEQ ID NO 6) or an enzyme having a sequence with at least 70% sequence identity; and

[0276] iv. Alternatively, Qs-28-O-XylT4 (SEQ ID NO 8) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 8.

[0277] 6. The method according to clause 2, wherein step a) comprises:

[0278] 1) Expression of genes required for the biosynthesis of QA-TriX into the host, and

[0279] 2) Introducing into the host a polynucleotide encoding:

[0280] i. Qs-28-O-FucT (SEQ ID NO 2) or an enzyme having a sequence with at least 70% sequence identity, alternatively QsFucSyn (SEQ ID NO 12) or an enzyme having a sequence with at least 45% sequence identity;

[0281] ii. Qs-28-O-RhaT (SEQ ID NO 4) or an enzyme having a sequence with at least 70% sequence identity;

[0282] iii. Qs-28-O-XylT3 (SEQ ID NO 6) or an enzyme having a sequence with at least 70% sequence identity; and

[0283] iv. Qs-28-O-ApiT4 (SEQ ID NO 10) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 10.

[0284] 7. A method according to any one of clauses 3 to 5, wherein step 1) comprises introducing into the host a polynucleotide encoding:

[0285] i. sapote 3-O-glucuronosyltransferase (QsCSL1, SEQ ID NO 26) or sapote 3-O-glucuronosyltransferase (QsCslG2, SEQ ID NO 28), or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 26 or 28;

[0286] ii. Quillaja saponaria QA-Monoβ-1,2-D-galactosyltransferase (Qs-3-O-GalT, SEQ ID NO 30) or an enzyme having a sequence with at least 70% sequence identity; and

[0287] iii. Quillaja saponaria QA-Di α-1,3-L-rhamnosyltransferase (DN20529_c0_g2_i8, SEQ ID NO 36), Quillaja saponaria QA-Di α-1,3-L-rhamnosyltransferase (Qs_0283850, SEQ ID NO 34) or Quillaja saponaria QA-Di dual β-1,3-D-xylosyltransferase / α-1,3-L-rhamnosyltransferase (Qs-3-O-RhaT / XylT, SEQ ID NO 32) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 36, 34 or 32, and / or Quillaja saponaria QA-Di β-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 having at least 70% sequence identity to SEQ ID NO 36, 34 or 32. 38 or 32 enzymes having a sequence with at least 70% sequence identity.

[0288] 8. A method according to any one of clauses 3 to 5, wherein step 1) comprises introducing into the host a polynucleotide encoding:

[0289] i. QsCSL1 (SEQ ID NO 26) or QsCslG2 (SEQ ID NO 28), or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 26 or 28;

[0290] ii. Qs-3-O-GalT (SEQ ID NO 30) or an enzyme having a sequence with at least 70% sequence identity; and

[0291] 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 having at least 70% sequence identity to SEQ ID NO 36, 34 or 32.

[0292] 9. The method according to any one of clauses 2 to 6, wherein step 1) further comprises introducing into the host a polynucleotide encoding:

[0293] i. QsCSL1 (SEQ ID NO 26) or QsCslG2 (SEQ ID NO 28), or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 26 or 28;

[0294] ii. Qs-3-O-GalT (SEQ ID NO 30) or an enzyme having a sequence with at least 70% sequence identity;

[0295] iii. Qs_0283870 (SEQ ID NO 38) or Qs-3-O-RhaT / XylT (SEQ ID NO 32) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 38 or 32.

[0296] 10. The method according to any one of clauses 2 to 9, wherein step a)-1) further comprises introducing into the host a polynucleotide encoding:

[0297] i. Quillaja β-amyrin synthase (QsbAS, SEQ ID NO 18) or an enzyme having a sequence having at least 50% sequence identity with SEQ ID NO 18;

[0298] ii. Quillaja scutellariae saponin C-28 oxidase (QsCYP716-C-28, SEQ ID NO 20), or an enzyme having a sequence having at least 50% sequence identity to SEQ ID NO 20;

[0299] iii. Quillaja scutellariae saponin C-16α oxidase (QsCYP716-C-16α, SEQ ID NO 22), or an enzyme having a sequence having at least 50% sequence identity with SEQ ID NO 22; and

[0300] iv. Quillaja scutellariae saponin C-23 oxidase (QsCYP714-C-23, SEQ ID NO 24), or an enzyme having a sequence having at least 50% sequence identity to SEQ ID NO 24.

[0301] 11. A method according to any one of clauses 2 to 10, wherein:

[0302] The amino acid SEQ ID NO 2 is encoded by the polynucleotide SEQ ID NO 1;

[0303] The amino acid SEQ ID NO 4 is encoded by the polynucleotide SEQ ID NO 3;

[0304] The amino acid SEQ ID NO 6 is encoded by the polynucleotide SEQ ID NO 5;

[0305] The amino acid SEQ ID NO 8 is encoded by the polynucleotide SEQ ID NO 7;

[0306] The amino acid SEQ ID NO 10 is encoded by the polynucleotide SEQ ID NO 9.

[0307] 12. A method according to any one of clauses 5 to 11, wherein:

[0308] The amino acid SEQ ID NO 26 is encoded by the polynucleotide SEQ ID NO 25;

[0309] The amino acid SEQ ID NO 28 is encoded by the polynucleotide SEQ ID NO 27;

[0310] The amino acid SEQ ID NO 30 is encoded by the polynucleotide SEQ ID NO 29;

[0311] The amino acid SEQ ID NO 32 is encoded by the polynucleotide SEQ ID NO 31;

[0312] The amino acid SEQ ID NO 34 is encoded by the polynucleotide SEQ ID NO 33;

[0313] The amino acid SEQ ID NO 36 is encoded by the polynucleotide SEQ ID NO 35;

[0314] The amino acid SEQ ID NO 38 is encoded by the polynucleotide SEQ ID NO 37.

[0315] 13. A method of making QA-Tri(X / R)-F*-GR-Ac, wherein an acetyl (Ac) moiety is attached to the C-4 position of the D-fucose of F*, a rhamnose (R) moiety is attached to the C-3 position of the D-fucose of F*, and a glucose (G) moiety is attached to the C-3 position of the rhamnose moiety of F*, wherein the method comprises combining QA-Tri(X / R)-F* with:

[0316] i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56;

[0317] ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64, or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and

[0318] 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 with SEQ ID NO 58.

[0319] 14. The method according to clause 13, wherein the method further comprises combining:

[0320] i. Qs-28-O-FucT (SEQ ID NO 2) or an enzyme having a sequence with at least 70% sequence identity, alternatively QsFucSyn (SEQ ID NO 12) or an enzyme having a sequence with at least 45% sequence identity to SEQ ID NO 12;

[0321] ii. Qs-28-O-RhaT (SEQ ID NO 4) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 4;

[0322] iii. Qs-28-O-XylT3 (SEQ ID NO 6) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 6; and

[0323] iv. Optionally, Qs-28-O-XylT4 (SEQ ID NO 8) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 8 and / or Qs-28-O-ApiT4 (SEQ ID NO 10) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 10.

[0324] 15. The method according to clause 13, wherein the method further comprises combining:

[0325] i. Qs-28-O-FucT (SEQ ID NO 2) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 2, alternatively QsFucSyn (SEQ ID NO 12) or an enzyme having a sequence having at least 45% sequence identity with SEQ ID NO 12;

[0326] ii. Qs-28-O-RhaT (SEQ ID NO 4) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 4;

[0327] iii. Qs-28-O-XylT3 (SEQ ID NO 6) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 6; and

[0328] iv. Optionally, Qs-28-O-ApiT4 (SEQ ID NO 10) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 10.

[0329] 16. The method according to clause 13, wherein the method further comprises combining:

[0330] i. Qs-28-O-FucT (SEQ ID NO 2) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 2, alternatively QsFucSyn (SEQ ID NO 12) or an enzyme having a sequence having at least 45% sequence identity with SEQ ID NO 12;

[0331] ii. Qs-28-O-RhaT (SEQ ID NO 4) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 4;

[0332] iii. Qs-28-O-XylT3 (SEQ ID NO 6) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 6; and

[0333] iv. Alternatively, Qs-28-O-XylT4 (SEQ ID NO 8) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 8.

[0334] 17. The method according to clause 13, wherein Tri(X / R) is TriX and F* is FRXA.

[0335] 18. A method according to any one of clauses 17, wherein the method further comprises combining:

[0336] i. Qs-28-O-FucT (SEQ ID NO 2) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 2, alternatively QsFucSyn (SEQ ID NO 12) or an enzyme having a sequence having at least 45% sequence identity with SEQ ID NO 12;

[0337] ii. Qs-28-O-RhaT (SEQ ID NO 4) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 4;

[0338] iii. Qs-28-O-XylT3 (SEQ ID NO 6) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 6; and

[0339] iv. Qs-28-O-ApiT4 (SEQ ID NO 10) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 10.

[0340] 19. A method according to any one of clauses 13 to 16, wherein the method further comprises combining:

[0341] i. QsCSL1 (SEQ ID NO 26) or QsCslG2 (SEQ ID NO 28), or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 26 or 28;

[0342] ii. Qs-3-O-GalT (SEQ ID NO 30) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 30; and

[0343] 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 having at least 70% sequence identity with SEQ ID NO 36, 34 or 32, and / or Qs_0283870 (SEQ ID NO 38) or Qs-3-O-RhaT / XylT (SEQ ID NO 32) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 38 or 32.

[0344] 20. A method according to any one of clauses 13 to 16, wherein the method further comprises combining:

[0345] i. QsCSL1 (SEQ ID NO 26) or QsCslG2 (SEQ ID NO 28), or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 26 or 28;

[0346] ii. Qs-3-O-GalT (SEQ ID NO 30) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 30; and

[0347] 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 having at least 70% sequence identity to SEQ ID NO 36, 34 or 32.

[0348] 21. A method according to any one of clauses 13 to 18, wherein the method further comprises combining:

[0349] i. QsCSL1 (SEQ ID NO 26) or QsCslG2 (SEQ ID NO 28), or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 26 or 28;

[0350] ii. Qs-3-O-GalT (SEQ ID NO 30) or an enzyme having a sequence having at least 70% sequence identity with SEQ ID NO 30; and / or

[0351] iii. Qs_0283870 (SEQ ID NO 38) or Qs-3-O-RhaT / XylT (SEQ ID NO 32) or an enzyme having a sequence having at least 70% sequence identity to SEQ ID NO 38 or 32.

[0352] 22. A method according to any one of clauses 13 to 21, wherein the method further comprises combining:

[0353] i. QsbAS (SEQ ID NO 18), an enzyme having a sequence having at least 50% sequence identity with SEQ ID NO 18;

[0354] ii. QsCYP716-C-28 (SEQ ID NO 20), an enzyme having a sequence having at least 50% sequence identity with SEQ ID NO 20;

[0355] iii. QsCYP716-C-16α (SEQ ID NO 22), an enzyme having a sequence having at least 50% sequence identity with SEQ ID NO 22; and

[0356] iv. QsCYP714-C-23 (SEQ ID NO 24), an enzyme having a sequence having at least 50% sequence identity with SEQ ID NO 24.

[0357] 23. A method according to any preceding clause, wherein the method comprises the step of isolating the QA-Tri(X / R)-F*-GR-Ac derivative.

[0358] 24. A method according to clause 23, wherein QA-Tri(X / R)-F*-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.

[0359] 25. The method of clause 24, wherein QA-Tri(X / R)-F*-GR-Ac is QA-TriX-FRXA-GR-Ac.

[0360] 26. QA-Tri(X / R)-F*-GR-Ac obtainable by the method of item 23.

[0361] 27. QA-Tri(X / R)-F*-GR-Ac according to clause 26, wherein QA-Tri(X / R)-F*-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.

[0362] 28. QA-Tri(X / R)-F*-GR-Ac according to clause 27, wherein QA-Tri(X / R)-F*-GR-Ac is QA-TriX-FRXA-GR-Ac.

[0363] abbreviation:

[0364] Apif: D-Apiofuranosyl

[0365] DMOT9: (3S,5S,6S)-3,5-dihydroxy-6-methyloctanoyl-CoA transferase 9

[0366] DN20529_c0_g2_i8: Quillaja sapodilla QA-Di α-1,3-L-rhamnosyltransferase

[0367] FR: disaccharide of β-D-fucose (F) and α-L-rhamnose (R) residues

[0368] FRX: trisaccharide of β-D-fucose (F), α-L-rhamnose (R), and β-D-xylose (X) residues

[0369] FRXX: a tetrasaccharide consisting of β-D-fucose (F), α-L-rhamnose (R), and two β-D-xylose (X,X) residues

[0370] FRXA: a tetrasaccharide of β-D-fucose (F), α-L-rhamnose (R), β-D-xylose (X), and β-D-apiose (A) residues

[0371] FRXX / A: FRXX or FRXA tetrasaccharide

[0372] Fucp: D-fucopyranose

[0373] FucSyn: an enzyme that promotes the production of fucosylated saponins

[0374] FSL:QsFucSyn-Like

[0375] Galp: D-galactopyranose

[0376] GlcpA: D-pyranoglucuronic acid

[0377] Glcp: D-glucopyranose

[0378] OS: 2,3-Oxysqualene

[0379] OSC: oxidosqualene cyclase

[0380] QA: Soap acid

[0381] QA derivatives

[0382] -QA-Di: 3-O-{β-D-galactopyranosyl-(1->2)-β-D-pyranoglucuronic acid}-saponin

[0383] -QA-Di-F: 3-O-{β-D-galactopyranosyl-(1->2)-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl fucosylate}-saponic acid

[0384] -QA-Di-FR: 3-O-{β-D-galactopyranosyl-(1->2)-β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1->2)-β-D-pyranoglucuronic acid}-saponic acid

[0385] -QA-Di-FRX: 3-O-{β-D-galactopyranosyl-(1->2)-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranoxylanosyl-(1->4)-α-L-pyranorhamnosyl-(1->2)-β-D-pyranoglucuronic acid}-saponin

[0386] -QA-Di-FRXA: 3-O-{β-D-galactopyranosyl-(1->2)-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-pyranoxylanosyl-(1->4)-α-L-pyranorhamnosyl-(1->2)-β-D-pyranoglucuronic acid}-saponin

[0387] -QA-Di-FRXX: 3-O-{β-D-galactopyranosyl-(1->2)-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1->3)-β-D-pyranosyl-(1->4)-α-L-pyranosyl-rhamnosyl-(1->2)-β-D-pyranoglucuronic acid}-saponin

[0388] -QA-Mono: 3-O-{β-D-pyranoglucuronic acid}-saponin

[0389] -QA-Mono-F: 3-O-{β-D-pyranoglucuronic acid}-28-O-{β-D-pyranoglucosyl ester}-saponic acid

[0390] -QA-Mono-FR: 3-O-{β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1->2)-β-D-pyranosylfucosylate}-saponic acid

[0391] -QA-Mono-FRX: 3-O-{β-D-pyranoglucuronic acid}-28-O-{β-D-pyranoxylanosyl-(1->4)-α-L-pyranorhamnosyl-(1->2)-β-D-pyranofucosyl ester}-saponic acid

[0392] -QA-Mono-FRXA: 3-O-{β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-pyranoxylanosyl-(1->4)-α-L-pyranorhamnosyl-(1->2)-β-D-pyranosylfucosylate}-saponic acid

[0393] -QA-Mono-FRXX: 3-O-{β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1->3)-β-D-pyranosyl-(1->4)-α-L-pyranosyl-rhamnosyl-(1->2)-β-D-pyranosylfucosylate}-saponic acid

[0394] -QA-TriR: 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-saponin

[0395] -QA-TriR-F: 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin

[0396] -QA-TriR-FR: 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0397] -QA-TriR-FRX: 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranoxylanosyl-(1->4)-α-L-pyranosyl-rhamnosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0398] -QA-TriR-FRXA: 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0399] -QA-TriR-FRXX: 3-O-{α-L-rhamnopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1->3)-β-D-pyranosyl-(1->4)-α-L-pyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0400] -QA-TriX: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-saponin

[0401] -QA-TriX-F: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-fucopyranosyl ester}-saponin

[0402] -QA-TriX-FR: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0403] -QA-TriX-FRX: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0404] -QA-TriX-FRXA: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0405] -QA-TriX-FRXX: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-xylopyranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin

[0406] -QA-TriX-G: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranoglucosyl ester}-saponic acid

[0407] -QA-TriX-GR: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{α-L-pyranosyl-(1->2)-β-D-pyranoglucuronic acid}-saponin

[0408] -QA-TriX-GRX: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-pyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-pyranoglucuronic acid}-saponin

[0409] -QA-Tri(X / R): QA glycosylated with a branched trisaccharide at the C-3 position, i.e. QA-TriX or QA-TriR

[0410] -QA-Tri(X / R)-F: QA glycosylated at C-28 and C-3, i.e. QA-TriX-F or QA-TriR-F

[0411] -QA-Tri(X / R)-FR: QA glycosylated at C-28 and C-3, i.e., QA-TriX-FR or QA-TriR-FR

[0412] -QA-Tri(X / R)-FRX: QA glycosylated at C-28 and C-3, i.e., QA-TriX-FRX or QA-TriR-FRX

[0413] -QA-Tri(X / R)-FRXA: QA glycosylated at C-28 and C-3, i.e., QA-TriX-FRXA or QA-TriR-FRXA

[0414] -QA-Tri(X / R)-FRXX: QA glycosylated at C-28 and C-3, i.e., QA-TriX-FRXX or QA-TriR-FRXX

[0415] -QA-Tri(X / R)-FRX(X / A): QA glycosylated at C-28 and C-3, i.e., QA-TriX-FRXX, QA-TriX-FRXA, QA-TriR-FRXX, or QA-TriR-FRXA

[0416] -QA-F: QA monoglycosylated at position C-28

[0417] -QA-FR: QA disaccharified at position C-28

[0418] -QA-FRX: QA triglycosylated at C-28

[0419] -QA-FRXA: QA tetraglycosylated at position C-28

[0420] -QA-FRXX: QA tetraglycosylated at position C-28

[0421] -QA-FRX(X / A): QA glycosylated at C-28, i.e. QA-FRXX or QA-FRXA

[0422] -QA-TriX-FRXA-Ac: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin acetylated at C-4 of D-fucose in C-28 chain

[0423] -QA-TriX-FRXA-R-Ac: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin, acetylated at C-4 of D-fucose of the core C-28 chain and with the rhamnose moiety attached to C-3 of D-fucose of the C-28 chain

[0424] -QA-TriX-FRXA-G: 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponin glycosylated at the C-3 position of the core C-28 rhamnose moiety

[0425] -QS-7 (or QA-TriX-FRXA-GR-Ac): 3-O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponic acid, which is acetylated at the C-4 position of the D-fucose of the core C-28 chain, and the rhamnose moiety is attached to the C-3 position of the D-fucose of the C-28 chain, and the glucose moiety is attached to the C-3 position of the rhamnose moiety of the core C-28 chain.

[0426] Qs_0283850: Quillaja sapodilla QA-Di α-1,3-L-rhamnosyltransferase

[0427] Qs_0283870: Quillaja sapodilla QA-Di β-1,3-D-xylosyltransferase

[0428] Qs-28-O-ApiT4: 28-O-fucosyl[1,2]-rhamnosyl[1,4]xylosyl[1,3] apiosyltransferase

[0429] Qs-28-O-FucT: 28-O-fucosyltransferase

[0430] Qs-28-O-RhaT: 28-O-fucosyltransferase

[0431] Qs-28-O-XylT3: 28-O-fucosyl[1,2]-rhamnosyl[1,4]xylosyltransferase

[0432] Qs-28-O-XylT4: 28-O-fucosyl[1,2]-rhamnosyl[1,4]xylosyl[1,3]xylosyltransferase

[0433] Qs-3-O-GalT: Quillaja sapodilla QA-Mono β-1,2-D-galactosyltransferase

[0434] Qs-3-O-RhaT: Quillaja sapodilla QA-Di α-1,3-L-rhamnosyltransferase

[0435] Qs-3-O-RhaT / XylT: Quillaja sylvestris QA-Di dual β-1,3-D-xylosyltransferase / α-1,3-L-rhamnosyltransferase

[0436] Qs-3-O-XylT: Quillaja sapodilla QA-Di β-1,3-D-xylosyltransferase

[0437] QS-7-AcetylT: 28-O-fucosidic acid [1,4] acetyltransferase (also known as QsACT-19')

[0438] QS-7-GlcT: 28-O-fucosyl[1,2]-rhamnosyl[1,3]glucosyltransferase (also known as QsUGT-BI)

[0439] QS-7-RhaT: 28-O-fucosyl[1,3]rhamnosyltransferase (also known as QsUGT-23500)

[0440] QsAXS1: UDP-D-Apiose / UDP-D-Xylose Synthase

[0441] QsACT-19': 28-O-fucosidic acid [1,4] acetyltransferase (also known as Qs-7-AcetylT)

[0442] QsbAS: Quillaja β-amyrin synthase

[0443] QsCSL1: Quillaja cellulose synthase-like enzyme (Quillaja cellulose 3-O-glucuronyltransferase)

[0444] QsCslG2: Quillaja cellulose synthase-like enzyme (Quillaja 3-O-glucuronyltransferase)

[0445] QsCYP716-C-28: Quillaja salsa odoratus C-28 oxidase

[0446] QsCYP716-C-16α: Quillaja salsa odoratus C-16α oxidase

[0447] QsCYP714-C-23: Quillaja salsa catechu acid C-23 oxidase

[0448] QsFSL-1: An enzyme from Quillaja saponaria that promotes the production of fucosylated saponins

[0449] QsFSL-2: An enzyme from Quillaja saponaria that promotes the production of fucosylated saponins

[0450] QsFucSyn: an enzyme from Quillaja saponaria that promotes the production of fucosylated saponins

[0451] QsUGT-BI: 28-O-fucosyl[1,2]-rhamnosyl[1,3]glucosyltransferase (also known as QS-7-GlcT)

[0452] QsUGT-0023500: Saponin 28-O-fucoside[1,3]rhamnosyltransferase (also known as QS-7-RhaT)

[0453] Rhap: L-rhamnose

[0454] SoFSL-1: an enzyme from S. officinalis that promotes the production of fucosylated saponins

[0455] UDP-sugar: uridine diphosphate sugar

[0456] UGT: UDP-dependent glycosyltransferase

[0457] Xylp: D-xylopyranose

[0458] refer to

[0459] Fleck JD, Betti AH, da Silva FP, Troian EA, Olivaro C, Ferreira F, VerzaSG. 2019. Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities. Molecules 24(1).

[0460] Kensil CR,Patel U,Lennick M,and Marciani D,1991.Separation and characterization of saponins with adjuvant activity from Quillaja saponariaMolina cortex,J Immunol.146(2)431-437.

[0461] Louveau T, Osbourn A. 2019. The Sweet Side of Plant-Specialized Metabolism. Cold Spring Harb Perspect Biol(In Press).

[0462] Reed J, Osbourn A. 2018. Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant Cell Reports.

[0463] 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.

[0464] 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.

[0465] 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.

[0466] P202303GB - Appendix A

[0467] SEQ ID NO 1: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 2.

[0468] ATGGAGAATGGGAGAGTTTACAAATCCCATGTCGTGGTGCTCGCATTTCACGGGCAAGGCCATATTGTTCCGTTAATCCAATTATCCAGACGATTGGCCTGGAAAGGCATCAAAATCACATTTGCTACAACACATTCTTGCACCAAGGCCATTCAAACAGGAAGTGATTCAATTTCACTTTTATCAATTTATGATGACATAACTGATGGTGGGTTTCAAGGAGAAGGAGGATTCAAGGGCTTCCTTCAGAGATTTGAAGCCAGTACCACAAGGATCTTACACGAATTCGTCAAGAATCATGAAAACTCAAAGAACCCAGTAAAATGCTTAATATATGATGCTAACTTAATATGGGCTCTGGAAATGGCAAAGCAATTGGGTATTGCTACTGCTGCATTTGTGTTTCCTTCTTGGGCTGCCATTGCCACCTACTATCCCTTTTATTTAGAGGTGTATGCGGATCAGCAGATAAAGAAGGTAGATCCTTTCACAATGCCTGACTTACCTCCACAACTTGGACTTCCAAATATGGCATCTCTCGGTTCAGATTCGGGTCAACACTCCCCCATACTCAAACTCATGTTGCAACAGTTAGAAAATTTTGGGAAAGCTGACTGGATCCTGTCTCACGCATTTGAACAGTTTGAACAAGAGGTACTTGACTGGATGAGAAATATCAGCCCAGTAACAACAATTGGTCCAACTCTGCCATCTGTTTATCTTGATGGTAGGCTAAAAGATGACACAGATTACGGTTACAATTTGTACAAGCCAGATAGTGATACCTGCATGAAGTGGCTAGACACTAAGGAAACTGAATCAGTGGTTTATATATCATTTGGCAGTGTTGCAGATTTGATCCCAGAACAGATGACAGAAATAACAAACTCCCTGAAGAAAATGAGCAGCAACTTTCTGTGGGTGGTGAAGGAAACTGAAAAAAACAACCTCCCTAGCAGCTTTGTTGAGGAGACAAAAGAAAAGGGATTGGTAGTGACTTGGTGCCCCCAGTTGAAGGTGTTGTCTCATCCTGCAGTGGGTTGTTTCATTACACACTGTGGAACAAATTCCATATTTGAGTCAGTATGCTTTGCAGTGCCAATGGTGGGAATGCCACAGTTTTGTGATCAAATGCCTAATGCATATTTCATGGAGAAGGTCTGGAAAGTAGGTGTTAGGCCAAGTTTGGATGACAATGGTGTCGTCACTGGAGAAGAAATTGAGCGATGTATAAAAGTAGTTACCGAAGGAGAGAGTGGGCAAGAGATTAAGAAGAAACTTGTGCAGTGGAAAGAGCTTGCAAAAGAGGCAGTGGACGAGGGTGGAAGTTCAGATAAGCACATTGATGAATTCATTGCTGGAATCACAACTTGA*

[0469] SEQ ID NO 2: Fucosyltransferase (Qs-28-O-FucT) capable of transferring β-D-fucopyranose to the C-28 position of quillaic acid.

[0470] MENGRVYKSHVVVLAFHGQGHIVPLIQLSRRLAWKGIKITFATTHSCTKAIQTGSDSISLLSIYDDITDGGFQGEGGFKGFLQRFEASTTRILHEFVKNHENSKNPVKCLIYDANLIWALEMAKQLGIATAAFVFPSWAAIATYYPFYLEVYADQQIKKVDPFTMPDLPPQLGLPNMASLGSDSGQHSPILKLMLQQLENFGKADWILSHAFEQFEQEVLDWMRNISPVTTIGPTLPSVYLDGRLKDDTDYGYNLYKPDSDTCMKWLDTKETESVVYISFGSVADLIPEQMTEITNSLKKMSSNFLWVVKETEKNNLPSSFVEETKEKGLVVTWCPQLKVLSHPAVGCFITHCGTNSIFESVCFAVPMVGMPQFCDQMPNAYFMEKVWKVGVRPSLDDNGVVTGEEIERCIKVVTEGESGQEIKKKLVQWKELAKEAVDEGGSSDKHIDEFIAGITT*

[0471] SEQ ID NO 3: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 4.

[0472]

[0473] SEQ ID NO 4: Rhamnosyltransferase capable of transferring α-1,2-L-rhamnose pyranose to QA-F (Qs-28-O-RhaT).

[0474] MAKTDKQLHIAMFPWLAMGHIFPNFELAKLFAQKGHSITLISTPRNISRLPQIPTHLEQLIKLVSLPILPKHKANLPENAESTMDVTPNKVPYLKMAYDGLQESLTQLLKSSAPDWIL YDFAADWLPPLVHSLQIRCVFFVVSPAWNLCFFDTPKPQLGSAAVFRTKPEDYLRPPSWVPFHSNIGLKLHEVKKMFEGVSDKETGVTVSFNFNKAVSSCDLFSFRSCYELESEWLNLV EDIYKRPVVPVGVIPPSFQVRIVNEEDNKPEWLKIQSWLDKQEQGSVVYIAFGSELKLGQQDLTELALGLELSGLPFFWALRKQQDSSSVDLPDGFEDRVSDRGVVCRDWVPQLKILA HGSIGGYLTHCGSGSVIEGLHFGRVLVMLPYLLDQALYARVLEEKKLGVEIPRNEQDGSFTRSSVAKSVKLAIVDEGGSIYRDKAKEMGLVFSDKDRHEQYIENFLQHLQHKREPFQI*

[0475] SEQ ID NO 5: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 6.

[0476]

[0477] SEQ ID NO 6: Xylosyltransferase capable of transferring β-1,4-D-xylopyranose to QA-FR (Qs-28-O-XylT3)

[0478] MAAAAPNHRLHIAFFPWLAFGHINPFFELAKLIAQKGHHISFISTPRNIQRLSQVPPQLADSIDLVSLPVIHNSNLPENAESTMDIPPDKTPYLGMLHDSLKEPLTQFLQTHSPDWILYDFSAGWLAAIVEDLGISHGYFSIIPCWNIGFNGRQMNGFQKPDISLPSAVSLKKYEVKK IMDLVKSFPKILDESATKSIASHSTCEVIFIRNCPEIEADWFDYTSKIFDKPVVPVGVVPPSVHITNKEKDEHFNKWLEIKEWLDQQDRGSVIYIAFGTESLPNQDEITMLAQGLELCGLPFFWALRKSNVASDQPNSDSVELPEGFEEQTKGRGIVWTSWAPQQRILGHNSIGGFVT HCGWSSVIEGIHYGRPLIMFPLTVEQSLNARILGEKKLGMEVPREDDGSFTGEVVAETLKLVLLDQDGKVYRDKVTEMSKVFGDKDKHEKYMGDLLEFFKNYRSLKRN*

[0479] SEQ ID NO 7: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 8.

[0480]

[0481] SEQ ID NO 8: Xylosyltransferase capable of transferring β-1,3-D-xylopyranose to QA-FRX (Qs-28-O-XylT4)

[0482] MDSTHLQPATTPLKIHFIPFISPGHIIPLSELARIFASRGEHVTIITTPFNADLLQKSIDEDRDSGEHIGIHTVEWSATELGLPHGIENLSNVTDLETAIKLHRALMLMQKQMEDFMTRNPPDCIIA DTFYPWASEFANRMGIPRLIFYPWSTFALCLMESIRSPDSPHRRLSSDSDPFVVPGLPHPIILTRSQLPEHDRKDIADPAAQLMDQHKETEMKSYGIILNNFAEIETEYTEHYKKITGHKVWHIGPAA AIVHRNAKEKAERVFKSDEHDNNLVINWLNSKEPNSVVYVCFGSGCQFPDKQLYEIACGLELSGHQFVWVVRGKDKQIDVNDDGEKTWLPKGFEERMKTENKGLIVRGWAPQVLVLDHPSLGCFLTH CGWNSTIEGITAGVPLITWPVFAEQFYNEKLITQVHGNGVVVGSEEWIMLFTVAKSLVSRDKIENAVRKIMDGGDEAVQIRRRARELGEKAWKAASTGGSSYNNLTAAIEDLKRLREDRSKLKTKTI*

[0483] SEQ ID NO 9: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 10.

[0484]

[0485] SEQ ID NO 10: Apitolysin glycosyltransferase (Qs-28-O-ApiT4) capable of transferring β-1,3-D-apiofuranosylose to QA-FRX.

[0486] MDSTHLQPATTPLKIHVIPFIAPGHIIPLSELARIFASRGEHVTIITTPFNADLLQKSIDEDRDSGEHIGIHTVEWSATELGLPHGVENLSNVTDLETGIKLHRALVLMQKQMEDFMTRNPPDCIIA DTFYPWASEFANRMGIPRLIFFPGCTFALCLMESIRSPDSPHRRLSSDSDPFVVPGLPHPIILTRSQLPEHDREDIADPAAQFMDQCKEAAMKSYGIILNNFAEIETEYTEHYKKITGHKVWHIGPA AAIVHRNAKEKAEGLFKSDEHDNNLVINWLNSKEPNSVVYVCFGSGCQFPDKQLYEIACGLELSGHQFIWVVRGKDKQIDVNDDEEKTWLPKGFEERMKTENKGLIVRGWAPQVLVLDHPSLGCFLT HCGWNSTIEGITAGVPLITWPVYSEQFYNEKLITQVHGNGVGVGSEEWIMLFSVAKSLVSRDKIENAVRKIMDGGDEALEIRRRARELGEKARKAASIGGSSDNNLTAAIEDLKRLREDRSKLKTKTI

[0487] SEQ ID NO 11: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 12.

[0488] ATGGCAGAAGCAACGCAGAGGTATGCTGTTGTGACAGGATCTAATAAGGGAATTGGATTTGGGATATGCAAGCAGTTGGCTTCTAAGGGAATCAAAGTAGTGTTAACAGCTAGAGATGAGAAGAGAGGTCTTGAAGCAGTTGAGAAATTGAAAGAAATTAGTCTGGCTGGTCATGTGGTTTTTCATCAACTCGATGTGTCTGATCCTGCTAGTGTTACTAGCCTTGAAGATTTCATCAAAACCCAGTTTGGGAAGCTAGATATTCTGGTAAACAATGCTGGGATAACAGGAACAACTGTAGATGCTGATGCTTTAGCAGCTTCAGGCTTCGGTACAGGGGGTGAACGTAAGCCTATTGATTGGAGTAAGTTAGTGATACAGACTTATGAATCAGTTGAAAAAGCTTTCAACACAAACTATTACGGTGGCAAAAGAATGACAGAAGCACTTATACCCCTCCTCCAGCTATCAGACTCACCCAGGATTGTTAATGTTTCCTCTGCTATGGGACAGTTAGAGAATATACCTAGTGGATGGGCAAAGGAAGTGCTCACAGATGTTGATAACCTAACAGAAGGAAAATTGGATGAGGTTTCAACCCAGTTTTTG AAAGATTTCAAAGAGGGTTCATTGGAAACCAAAGGCTGGCCTAGTCTTATGTCTTCTTATATAGTCTCAAAAGCTGTTTTAAATGCCTACACAAGGATTCTTGCTAAGAAATACCCAGCTTTCTGCATCAATTGTGTAGATCCTGGCTATGTGAAGACAGACATAAACCATCATACTGGCCAATTAAGTGTTGATGAAGGTGCTGAAAGTCCTGTAAGACTGGCCTTGCTGCCTAATGGTGGTCCTTCTGGCGTGTTCTTCTCCAGGACAGAAGAAGCACCATTTTGA

[0489] SEQ ID NO 12: Reductase (QsFucSyn) capable of enhancing the activity of fucosyltransferase

[0490] MAEATQRYAVVTGSNKGIGFGICKQLASKGIKVVLTARDEKRGLEAVEKLKEISLAGHVVFHQLDVSDPASVTSLEDFIKTQFGKLDILVNNAGITGTTVDADALAASGFGTGGERKPIDWSKLVIQTYESVEKAFNTNYYGGKRMTEALIPLLQLSDSPRIVNVSSAMGQLENIPSGWAKEVLTDVDNLTEGKLDEVSTQFLKDFKEGSLETKGWPSLMSSYIVSKAVLNAYTRILAKKYPAFCINCVDPGYVKTDINHHTGQLSVDEGAESPVRLALLPNGGPSGVFFSRTEEAPF*

[0491] SEQ ID NO 13: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 14.

[0492]

[0493] SEQ ID NO 14: Enzyme capable of enhancing the activity of celery glycosyltransferase (QsAXS1).

[0494] MPTLYKKAGLMASASRVDLDGNQIKPMTICMIGAGGFIGSHLCEKIMAETPHKVLALDVYNDKIKHLLEPDSLQWKDRIQFHRINIKHDSRLEGLIKMA DLTINLAAICTPADYNTRPLDTIYSNFIDALPVVKYCSENNKRLIHFSTCEVYGKTIGSFLPKDSPLLKDPEYFVLKEDASPCIFGPIEKQRWSYACAKQ LIERLVYAEGAENGLEFTIVRPFNWIGPRMDFIPGIDGPSEGVPRVLACFSNNLLRGEPLKLVDGGQSQRTFVYIKDAIEAVLLMIENPARANGHIFNVGNPHNEVTVRQLAEMMTEVYSKVSSGEPSLEVPTIDVSSKEFYGEGYDDSDKRIPDMTIINRQLGWNPKTSLWDLLESTLTYQHRTYAEAIKKSIAKPVAS*

[0495] SEQ ID NO 15: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 16.

[0496]

[0497] SEQ ID NO 16: AstHMGR (Avena strigosa truncated HMG-CoA reductase) translated nucleotide sequence (424 aa):

[0498] MAPEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDGFDYDSILGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCL IASTNRGCKAIAESGGASSVVYRDGMTRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFTCSTGDAMGMNMVSKGVQNVL DYLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSVVCEAVIREEVVHKVLKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVES SQCITMLEAVNDGRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVLAGELSLISAQAAGHLVQSHMKYNRSSKDMSKIAC

[0499] SEQ ID NO 17: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 18.

[0500]

[0501] SEQ ID NO 18: Enzyme (QsbAS) involved in the production of β-amyrin from 2,3-oxidosqualene

[0502] MWRLKIAEGGSDPYLFSTNNFVGRQTWEFEPEAGTPEERAEVEAARQNFYNNRYQVKPCDDLLWRYQFLREKNFKQTIPPVKVEDGQEITYEMATTSMQRAARHLSALQASDGHWPAQIAGPLFFMPPLVFCVYITGHLNTVFPSEHRKEILRYMYYHQNEDGGWGLHIEGHSTMFCTALNYICMRILGEGPEGGQDNACARARMWILDHGGVTHIPSWGKTWLSILGLFEWSGSNPMPPEFWILPSFLPMHPAKMWCYCRMVYMPMSYLYGKRFVGPITPLIVQLREEIHTQNYHEINWKSVRHLCAKEDIYYPHPLIQDLIWDSLYILTEPLLTRWPLNKLVRERALQVTMKHIHYEDENSRYITIGCVEKVLCMLACWVDDPNGDAFKKHLARVPDYVWVSEDGITMQSFGSQEWDAGFAVQALLASNLTEELGPALAKGHDFIKQSQVKDNPSGDFKSMYRHISRGSWTFSDQDHGWQVSDCTAEGLKCCLLLSMLPPEIVGEKMEPQRLFDSVNVLLSLQSKKGGLAAWEPAGAQDWLELLNPTEFFADIVVEHEYVECTGSAIQALVLFKKLYPGHRKKEIDSFITNAVRFLENTQTADGSWYGNWGVCFTYGCWFALGGLAAAGKTYNNCPAIRKAVNFLLTTQREDGGWGESYLSSPKKIYVPLEGSRSNVVHTAWAMMGLIHAGQAERDSTPLHRAAKLIINYQLENGDWPQQEITGVFMKNCMLHYPMYRNIYPMWALAEYRRRVPLP*

[0503] SEQ ID NO 19: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 20

[0504]

[0505] SEQ ID NO 20: Enzyme involved in the production of oleanolic acid from β-amyrin (QsCYP716-C-28)

[0506] MEHLYLSLVLLFVSSISLSLFLFYKHKSMFTGANLPPGKIGYPLIGESLEFLSTGWKGHPEKFIFDRMSKYSSQIFKTSILGEPTAVFPGAVCNKFLFSNENKLVNAWWPASVDKIFPS SLQTSSKEEAKKMRKLLPQFLKPEALHRYIGIMDSIAQRHFADSWENKNQVIVFPLAKRYTFWLACRLFISVEDPTHVSRFADPFQLLAAGIISIPIDLPGTPFRKAINASQFIRKELLA IIRQRKIDLGEGKASPTQDILSHMLLTCDENGQYMNELDIADKILGLLVGGHDTASAACTFIVKFLAELPHIYEQVYKEQMEIAKSKVPGELLNWEDIQKMKYSWNVACEVMRLAPPLQG AFREAITDFVFNGFSIPKGWKLYWSANSTHKSPDYFPEPDKFDPTRFEGNGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKLVPDEKIVVDPMPIPAKGLPVRLYPHKA*

[0507] SEQ ID NO 21: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 22.

[0508] ATGATATATAATAATGATAGTAATGATAATGAATTAGTAATCAGCTCAGTTCAGCAACCATCCATGGATCCTTTCTTCATTTTTGGCTTACTTCTCTTGGCTCTCTTTCTCTCTGTTTCTTTTCTTCTCTACCTTTCCCGTAGAGCCTATGCTTCTCTCCCCAACCCTCCGCCGGGGAAGCTCGGCTTCCCCGTCGTCGGCGAGAGTCTCGAATTTCTCTCCACCCGACGCAAAGGTGTTCCTGAGAAATTCGTCTTCGACAGAATGGCCAAATACTGTCGGGATGTCTTTAAGACATCAATATTGGGAGCAACCACCGCCGTCATGTGCGGCACCGCCGGTAACAAATTCTTGTTCTCCAACGAGAAAAAACACGTCACTGGTTGGTGGCCGAAATCTGTAGAGCTGATTTTCCCAACCTCACTTGAGAAATCATCCAACGAAGAATCCATCATGATGAAACAATTCCTTCCCAACTTCTTGAAACCAGAACCTTTGCAGAAGTACATACCCGTTATGGACATAATTACCCAAAGACACTTCAATACAAGCTGGGAAGGACGCAACGTGGTCAAAGTGTTTCCTACGGCTGCCGAATTCACCACGTTGCTGGCTTGTCGGGTATTCCTCAGTGTTGAGGATCCCATTGAAGTAGCCAAGATTTCAGAGCCATTTGAAATCTTAGCTGCTGGGTTTCTTTCAATACCCATAAATCTTCCGGGTACCAAATTAAATAAAGCGGTTAAGGCAGCGGATCAGATTAGAGACGCAATTGTACAGATTTTGAAACGGAGAAGGGTTGAAATTGCGGAGAATAAAGCAAATGGAATGCAAGATATAGCGTCCATGTTGTTGACGACACCAACTAATGCTGGGTTTTATATGACCGAGGCTCACATTTCTGAGAAAATTTTGGGTATGATTGTTGGTGGCCGTGATACTGCTAGTACTGTTATCACCTTCATCATCAAGTATTTGGCAGAGAATCCTGAAATTTATAATAAGGTCTATGAGGAGCAAATGGAAGTGGTAAAGTCAAAGAAACCAGGTGAGTTGCTGAACTGGGAAGATGTGCAGAAAATGAAGTACTCTTGGTGCGTAGCATGTGAAGCTATGCGACTTGCTCCTCCTGTTCAAGGTGGTTTCAAGGTGGCCATTAATGACTTTGTGTATTCTGGGTTCAACATTCGCAAGGGTTGGAAGTTATATTGGAGTGCCATTGCAACACACATGAATCCAGAATATTTCCCAGAACCTGAGAAATTCAACCCCTCAAGGTTTGAAGGGAAGGGACCAGTACCTTACAGCTTCGTACCCTTCGGAGGCGGACCTCGGATGTGTCCCGGGAAAGAGTATTCCCGGCTGGAAACACTTGTTTTCATGCATCATTTGGTGACGAGGTACAATTGGGAGAAAGTGTATCCCACAGAGAAGATAACAGTGGATCCAATGCCATTCCCTGTCAACGGCCTCCCCATTCGCCTTATTCCTCACAAGCACCAATGA

[0509] SEQ ID NO 22: Enzyme (QsCYP716-C-16α) involved in the production of euscaphic acid from oleanolic acid

[0510] MIYNNDSNDNELVISSVQQPSMDPFFIFGLLLLALFLSVSFLLYLSRRAYASLPNPPPGKLGFPVVGESLEFLSTRRKGVPEKFVFDRMAKYCRDVFKTSILGATTAVMCGTAGNKFLFSNEKKHVTGWWPKSVELIFPTSLEKSSNEESIMMKQFLPNFLKPEPLQKYIPVMDIITQRHFNTSWEGRNVVKVFPTAAEFTTLLACRVFLSVEDPIEVAKISEPFEILAAGFLSIPINLPGTKLNKAVKAADQIRDAIVQILKRRRVEIAENKANGMQDIASMLLTTPTNAGFYMTEAHISEKILGMIVGGRDTASTVITFIIKYLAENPEIYNKVYEEQMEVVKSKKPGELLNWEDVQKMKYSWCVACEAMRLAPPVQGGFKVAINDFVYSGFNIRKGWKLYWSAIATHMNPEYFPEPEKFNPSRFEGKGPVPYSFVPFGGGPRMCPGKEYSRLETLVFMHHLVTRYNWEKVYPTEKITVDPMPFPVNGLPIRLIPHKHQ*

[0511] SEQ ID NO 23: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 24.

[0512]

[0513] SEQ ID NO 24: Enzyme (QsCYP714-C-23) involved in the production of quillaic acid from euscaphic acid.

[0514] MWFTVGLVLVFALFIRLYSSLWLKPRATRIKLSNQGIKGPKPAFLLGNVAEMRRFQSKLPKSELKQGQVSHDWASKSLFPFFSLWSQKYGNTFVFSLGNIQVLYVSDHELVKEINQNTSLDLGKPKYLQKERGPLLGQGILTSNGQLWAYQRKIMTPELYKEKIKGMCELMVESVAWLVEEWGTKIQAEGGAADIRIDEDLRSFSGDVISKACFGSCYAGGREIFLRLRALQHQIASKALLMGFPGLKYLPIKSNREIWRLEKEIFQLIMKLAEDRKKEQHERDLLQIIIEGAKSSDLSSEAMAKFIVDNCKNVYLAGHETTAMSAGWTLLLLANHPEWQARVRDEILQVTEGRNPDFDMLHKMKLLTMVIQEALRLYPTVIFMSREALEDINVGNIQVPKGVNIWIPVVNLQRDTTVWGADANEFNPERFANGVNNSCKVPQLYLPFGAGPRICPGINLAMTEIKILLCILLTKFSFSVSPNYRHSPVFKLVLEPENGINVIMKKL*

[0515] SEQ ID NO 25: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 26.

[0516]

[0517] SEQ ID NO 26: Enzyme (QsCSL1) involved in the production of QA-mono from saponarin.

[0518] MKSPSNPNQKPILHTCTIQQPRATLNKIHSLIHFSAILVLFYYRITRLFFTDDFKVPKLLWTLMTISEFILAFIWVLIQPFRWRPVSRSVIPENMPKDISLPAVDVFVCTADPQKEPTVEVMNTILSAMALDYPAEKLAVYLSDDGGSAVTLYAIKEACCFAKMWLPFCNKYGIKSRCPEAYFSKLAADEWLHRSVEFVAEEKEVKANYEEFKRNVQKFGEQQENSRVVHDRHPHVEIIHNNWNNEDQAHEMPLLVYVSRERRPSHHPRFKAGALNTLLRVSGIISNSPYILVLDCDMYCNDPTSARQAMCFHLDPQLSKNLAFVQFPQIFYNASKNDVYDAQVRAAYQTKWQGMDGLQGPIFSGTGFYLKRKAMYGNPDQDDNCLLKPYKKFGMSGEFVESLKVLNEQDGTQKKLLDGFLQEAKLLASCAYETKTSWGKEIGFSYDCLIESTFTGYLLHCRGWISVYLYPKRPCFLGCCPTDMKDAMVQYTKWMSELFSIAISRFNPLLYGVARMSILQSLCYGSFTLAPILSFPLFLYGTVPQLCLLKGISLFPKVSDPWFAVFAAIFVSSLCQHWFEVLSCDGTFTTWCNEQRSWLIKSVSGSLFGVVGAILQRLGLKTKFSLSNKAMDKEKLEKYEKGKFNFQGAAMFMVPVSILVILNTFCFLGGFWKVIIMKNILDMFGQLSLSAYVLVLSCPVLEGMLTRISKKMV*

[0519] SEQ ID NO 27: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 28.

[0520]

[0521] SEQ ID NO 28: Enzyme (QsCslG2) involved in the production of QA-mono from saponarin.

[0522] MATVSSLHTCTVQQPRAAINRIHIFLHFIAILFLFYYRVTGLFYDNAVPTLAWSLMTLAELIFAFVWVLSQAFRWRPVLRSVIPERIPKDVRLPAVDILICTADPLKEPTVEVMNTVLSAMALDYPAENLAVYLSDDGGSPVTLFAMKQVGPFAKLWLPFCNKYGIKTRHPESFFSAFADDERLHRSDEFRAEEEAIKDKYEEFKRTIEKYGGEGKNSHVVQDRPPHVEIIHDTRKIRENSEDQAVPLLVYVSREKRPSYNSRFKAGALNTLLRVSGVISNSPYVLVLDCDMYCNDPTSARQAMCFHLDPQMSRTLSFVQFPQVFYNVSKNDIYDGQARAAFKTKWQGMDGLRGPLLSGTGFYLKRKSLYGSPNQEDDCLLEPHKNFGKCDKLIESVKVIYERDVSIKADSSDAILQDAKQLASCPYETNTSWGKEVGFSYDCLLESTFTGYLLHCRGWTSVYLYPKKPCFLGCTPVDMKEAMVQYTKWISELFLLAISRFNPLTFGISRMSILQSMCYGYLTIMPILSVAMIFYATVPQLCLLRGVPLFPKVSDPWFAVFLAIFVSSLCQHLIEVLTSDGTLKTWWNEQRNWVIKSGSGSVFGALSGILKWFGMKIKFGLSNKAVDKEKLEKYEKGKFDFQGAAMFMVPLTISVILNTLCLIGGLWRVITLKNFEEMSGQFIISLYFLALSYPILEGLLRKGKGKA

[0523] SEQ ID NO 29: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 30.

[0524] ATGGTGGAGTCTCCAGCAGATCATGATGTGCTCAAAATCATTGTCCTTCCATGGGTAACCTCAGGTCACATGATTCCCATGGTAGATGCAGCCAGACTATTTGCTATGCATGGTGCAGATGTTACCATCATCACCACCCCAGCTAATGCCCTTACATTCCAGAAATCCGTCGACCGTGATTTCAATTCCGGTCGTTTAATCAGAACTCACACCCTTAAATTCCCTGCAGCAGAAGTTGGTGTACCTGAAGGAGTTGAAAACTTCAACAATACTTCCCCTGAAATGACCTCCAAAGTCTACCTTGGAGTCTCAATGCTCCGAGAACCAACCCAACAATTGATTGAGGATCTGCGTCCAGATTGTCTTATCACTGATATGTTCTATCCTTGGGCTGTGGATGTTGCTGACAAATTAGGCATTCCAAGGCTAATTTTTCAAGGTCCTGGAAGTTTTGGTTTGTCAGCTATGCATTCTATCAAACAGTATGAGCCCTTTAAGTCAGTAACTTCAGATACTGAGACATTCCCACTACCTGGATTGCCGCATAAGGTAGAGATGACAAGGTTGCAGATACCAAAATGGGTTCGTGAGCCAAATGGGTACACTCAATTGATGGGCAGGGTAAAAGATTCGGAGAGAAGAAGCTATGGGTCATTGGTGAATAGCTTTTATGACTTCGAAGGCCCTTATGAAGAGCACTATAGGAAGGCAACAGGACAGAGGGTTTGGAGCATTGGACCAGTTTCAGTTTGGGTGAACCAAGATGCTGCAGATAAGGTTGGAAGAGGACAGGATCTTGTTGCTGAAGACCAAAACAGCTGGTTGAATTGGCTCAATTCCAAAGAGAAAAACTCTGTTCTGTATGTAAGTTTTGGGAGCATGGCCAAGTTCCCATCTGCTCAGCTTCTTGAAATAGCTCATGGGCTTGAAGCTTCAGGTCATAGTTTCATCTGGGTTGTCAGAAAAGTTGACGGGGATGATGATGTAGACGTGTGGCTTCCAGATTTTGAGAAGAAAATGAAAGAGAACAACAAGGGTTTCATCATAAGGAATTGGGCACCACAATTGCTCATATTGGACCATCCAGCAATTGGAGGTTTGCTGAATCACAGTGGATGGAATTCAGTACTGGAAGGTGCTACAGCAGGCTTGCCAATGATCACTTGGCCTCTGTATGCCGAGCATTTTTACAATGAAAGGTTGGTTCTAGATGTGTTGAAAATTGGAGTACCAGTTGGGGTGAAGGAGTGGAAGAACTTGCATGAGGTGGGTGAGTTGGTGAGAAGGGATGCAATTGCCAAGGCAATTAAATTGTTAATGGGTAGTGGAGAAGAAGCTGAGGTAATGAGGAAAAAAGCCAAAGAGCTTGGTGTTGGAGCAAAGAAAGGTATTCAGGTTGGAGGTTCTTCTCATACCAATTTGATAGCAGTGATTGATGAGTTAAAGTCACTAAAGAAATCAAGAATTCAGGGTGTCTGA

[0525] SEQ ID NO 30: Enzyme (Qs-3-O-GalT) involved in the production of QA-Di from QA-Mono.

[0526] MVESPADHDVLKIIVLPWVTSGHMIPMVDAARLFAMHGADVTIITTPANALTFQKSVDRDFNSGRLIRTHTLKFPAAEVGVPEGVENFNNTSPEMTSKVYLGVSMLREPTQQLIEDLRPDCLITDMFYPWAVDVADKLGIPRLIFQGPGSFGLSAMHSIKQYEPFKSVTSDTETFPLPGLPHKVEMTRLQIPKWVREPNGYTQLMGRVKDSERRSYGSLVNSFYDFEGPYEEHYRKATGQRVWSIGPVSVWVNQDAADKVGRGQDLVAEDQNSWLNWLNSKEKNSVLYVSFGSMAKFPSAQLLEIAHGLEASGHSFIWVVRKVDGDDDVDVWLPDFEKKMKENNKGFIIRNWAPQLLILDHPAIGGLLNHSGWNSVLEGATAGLPMITWPLYAEHFYNERLVLDVLKIGVPVGVKEWKNLHEVGELVRRDAIAKAIKLLMGSGEEAEVMRKKAKELGVGAKKGIQVGGSSHTNLIAVIDELKSLKKSRIQGV*

[0527] SEQ ID NO 31: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 32.

[0528]

[0529] SEQ ID NO 32: Enzyme (Qs-3-O-RhaT / XylT) involved in the production of QA-TriR or QA-TriX from QA-Di.

[0530] MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASGYHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIVADMFTSWTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPDGTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLIHRNDEDKIQRSHKTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDEDNDDVGNKWLPAFEERIKKENKGMILRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKAAQDHES*

[0531] SEQ ID NO 33: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 34.

[0532]

[0533] SEQ ID NO 34: Enzyme (Qs_0283850) involved in the production of QA-TriR from QA-Di.

[0534] MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKGKTSGYHVNIHAVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPDCIVADMFNRWTSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDGAFSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGPLQLIYQNDDPKVQRSQKTAVVSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVGNKWLPDFEERIKRENKGIIFRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDCGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKVA*

[0535] SEQ ID NO 35: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 36.

[0536]

[0537] SEQ ID NO 36: Enzyme involved in the production of QA-TriR from QA-Di (DN20529_c0_g2_i8).

[0538] MVSGDDTVSRPLIVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKRKNSGYHVNIHTVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPDCIVADMFNRWTSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDGAFSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGPLQLIYQNDDPKVQRSQKTAVLSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVGNKWLPGFEERIKRENKGIIFRGWAP QMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRAREMKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKAAMKS*

[0539] SEQ ID NO 37: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 38.

[0540]

[0541] SEQ ID NO 38: Enzyme involved in the production of QA-TriX from QA-Di (Qs_0283870).

[0542] MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASGYHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIV ADMFTSWTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPDGTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLI HRNDEDKIQRSHKTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDEDNDDVGNKWLPAFEERIKKENKGMILRGWAPQMLILNHPAIGGFMTHCG WNAAVEALSSGVPIITFPVFSDQFYNERLISQVHKCGVGVGTEAWSYAFDAGKNPVGREKIMTAVKKILDGGEEAEGMRKRARELKEIAKRSVEEGGSSYNNLTAMIQDLKEFRANNGKAAQDHES*

[0543] SEQ ID NO 39: Acanthocystis turfacea chlorella virus 1 UDP-D-glucose 4,6-dehydratase (ATCV-1) coding sequence (1053 bp):

[0544] NB: This sequence was codon optimized for expression in Nicotiana benthamiana. The original sequence can be found in Genbank ID: NC_008724.1 (see locus tag ATCV_z554R).

[0545]

[0546] SEQ ID NO 40: Translated nucleotide sequence of Cnidaria Chlorella virus 1 UDP-D-glucose 4,6-dehydratase (ATCV-1) (350 aa):

[0547] MNSQEYTPKSVLVTGGAGFIGSHVVMKLVQRYPECKVVVLDKMDYCATLNNLATVRDAPNFKFVKGDIQSTDLLAHVLKQEKIDTIMHFAAQTHVDNSFGNSLAFTMNNVYGTHVLLECARLYGGVQRFINVSTDEVYGESSLGKKEGLDEHSSLEPTNPYAAAKAGAEMMARAY HTSYKLPIVTRGNNVYGPHQFPEKMIPKFILRATRGLDLPIHGDGGALRSYLYVDDVAEAYITILLKGNVGETYNIGTQKERSVVDVAHDICKIFNRDSDTAIWHVKDRAFNDRRYFISDKKLLDLGWQEKTTWEDGLKQTVGWYLQHATRSYWDHGNMELALDAHPTLQVPKF*

[0548] SEQ ID NO 41: Aggregatibacter actinomycetemcomitans NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AaFCD) coding sequence (693 bp):

[0549] NB: This sequence has been codon-optimized for expression in Nicotiana benthamiana. The original sequence can be found in Genbank ID: Ab002668.1 (sequence 15271..15963 bp).

[0550] ATGATAATTGGCAACGGTATGCTGGCAAAAGCCTTTGAATCATTCCATAAGCGAACTTACAATTACATAATATTTGCATCCGGAGTGAGCAACTCAAACGAAACTTCCTTCGAGAATTTCAACAGAGAGAAGGAATTGCTTCTTGAAGTCCTGGAGCAATATAAAGACAAAACTATCGTTTACTTTAGTTCCTGCTCCATATACGATTCTAGTTTGACGAATTCTTTGTATGTCTACCACAAAATGTGTATGGAGAGACTGGTGCGTGAAAACTCCAAGAATTATCTCATAGCCCGTCTCCCCCAAGTTATTGGTAAAACGTATTCACCAACCATTGTCAACTTCCTTTTTAACAAAATCAAAAATAGGGAGTGTTTCAGCATATTCGGAAAAGCTCACCGAAATTTTATCGACGTGGATGATGTCGTTAAGGTCACCAATTACTTATTGAAGGAGGGTCTGTTCATTAACAGTATTGTGAACTTGGCAAGCACGCACCATACCTCCATGTACGAATTAATCTTATATTTGGAAAAAATAAGTAATCAACGTGCCTTCTATAATGTTGAGAACAAAGGGTCTAGGTACTTTATTGATGTTTCAATACTGCAGGATGTTTATCAGAAGCTGGGGATCAAATTTGACAAAGATTACGTAGAAAAGGTTATCAACAAGTACTACGCTATTAAGTAA

[0551] SEQ ID NO 42: Nucleotide sequence (230 aa) translated from NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AaFCD) of Aggregatibacter actinomycetemcomitans

[0552] MIIGNGMLAKAFESFHKRTYNYIIFASGVSNSNETSFENFNREKELLLEVLEQYKDKTIVYFSSCSIYDSSLTNSLYVYHKMCMERLVRENSKNYLIARLPQVIGKTYSPTIVNF LFNKIKNRECFSIFGKAHRNFIDVDDVVKVTNYLLKEGLFINSIVNLASTHHTSMYELILYLEKISNQRAFYNVENKGSRYFIDVSILQDVYQKLGIKFDKDYVEKVINKYYAIK*

[0553] SEQ ID NO 43: Anoxybacillus tepidamans NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AtFCD) coding sequence (927 bp):

[0554] NB: This sequence has been codon-optimized for expression in Nicotiana benthamiana. The original sequence can be found in Genbank ID: AY883421.5 (sequence 13209-14135 bp).

[0555] ATGAAGAGGATACTGATACTCGGATGCGGTTACCTGGGTTTAAATCTCGCAAACTATTTTTGTAAAAAAAATTATGATGTCTCAGTGATAGGGAGAAAGTCTGTCTATAGCAATTTTTTGGAAGAGGAGATAGAGTTCATAGAAGATGATATCAAAAATATAAATAGTTATAAGCACATGTTTAATGAGGAGACAACCGTCATTTACGCCATAGGAAGTATTAACGCAAATAACTATTTTATGGACCTGAGGAATGATATAGAAAACTCATACATCCCCTTCATTAACCTCCTTAACTTTCTTTCCGAAAAGTATATTCAAAAGTTCGTCTTTCTCTCTTCAGCCGGAACAGTCTATGGGAACGTGAATAAGAATTATATAAGCGAGAATGAGATTCTTAACCCAATTTCAATCTATGGTTTGCAGAAAGCCTTCTTTGAACAACTGATAAGGATTAAAAACA ATGAGGCTAGCCATTTCAGGTATTTGATCTTCAGAATATCTAACCCCTATGGGGGAATCAACATTCCGAACAAGAATCAGGGAATTATTCCGACGTTAGTGTACAAAGCCGTGAACAATGAGCCTTTCGAACTTTGGGCATCAATCAATACCATCCGTGATTATATATTTACATCGATGACCTTAGCGAATTGATCTACAAAACAATCTATCTGGACATTTATAACGAGACCCT CAATCTCGGGTCCGGTAAAGGAACATCAATCAAGCAACTCATTAGCCTCGTGGAGGAGATTTTGGGAAAGAAGATCACTATTCTTGAAAAGCCCCCCATAAAGACTAACGTTTTGAAAAATATACTTGATATTTCTAAGCTCGTCAACACCGTAGGCTACGAACCAAAGATCAGCATTGAAGAGGGTATTAGCCGTTACATCAACACTATTTTTAACGAAGAACATTTTTTAA

[0556] SEQ ID NO 44: Thermophilic anaerobes NDP-4-keto-6-deoxy-glucose 4-ketoreductase (AtFC) translated nucleotide sequence (308aa):

[0557] MKRILILGCGYLGLNLANYFCKKNYDVSVIGRKSVYSNFLEEEIEFIEDDIKNINSYKHMFNEETTVIYAIGSINANNYFMDLRNDIENSYIPFINLLNFLSEKYIQKFVFLSSAGTVYGNVNKNYISENEILNPISIYGLQKAFFEQLIRIKN NEASHFRYLIFRISNPYGGINIPNKNQGIIPTLVYKAVNNEPFELWASINTIRDYIYIDDLSELIYKTIYLDIYNETLNLGSGKGTSIKQLISLVEEILGKKITILEKPPIKTNVLKNILDISKLVNTVGYEPKISIEEGISRYINTILTKNIF*

[0558] SEQ ID NO 45: Escherichia coli NDP-4-keto-6-deoxy-glucose 4-ketoreductase (EcFCD) coding sequence (951 bp):

[0559] NB: This sequence has been codon-optimized for expression in Nicotiana benthamiana. The original sequence can be found in Genbank ID: AY528413.1 (sequence 3156-4106 bp).

[0560] ATGGATGCTCGTAAAAATGGGGTATTAATAACCGGTGGAGCTGGGTTCATAGGTAAAGCCTTAATAACTGAAATGGTCGAACGTCAAATTCCCCTGGTGTCATTTGACATCAGCGATAAGCCCGACAGTTTGCCAGAGCTTTCCGAATATTTCAACTGGTATAAATTCTCATACCTTGAGAGTTCACAGAGGATTAAAGAGCTTCACGAAATAGTTTCCAGGCATAACATCAAAACGGTCATCCATTTAGCTACAACTATGTTTCCCCACGAATCCAAAAAGAACATCGATAAGGATTGCTTAGAAAACGTTTATGCCAACGTGTGTTTCTTTAAGAATTT ATATGAAAACGGCTGTGAAAAAATTATCTTCGCCTCATCAGGTGGCACCGTATATGGGAAGTCTGATACACCCTTCTCCGAAGACGATGCCCTGCTTCCCGAAATTTCCTACGGACTGTCCAAGGTTATGACTGAAACTTATCTCCGATTCATAGCCAAGGAATTGAATGGGAAGTCCATCTCTCTCAGAATATCTAACCCCTATGGTGAGGGGCAAAGGATTGACGGGAAACAAGGAGTCATTCCAATTTTCCTCAATAAAATCAGCAACGACATCCCCATCGACATCATTGGCTCTATCGAATCAAAGCGAGACTACATTTATATTTCAGATCTCGTACAAGCTTTCATGTGCTCTCTGGAATATGAAGGTCACGAAGACATATTTAATATAGGTTCTGGGGAAAGCATAACTCTGAAGAAATTGATCGAGACGATTGAGTTCAAGCTGAACAAGAAGGCTGTGATTGGATTTCAAGATCCGATCCACACCAATGCCAATGGTATAATTCTCGACATCAAACGAGCCATGGCAGAACTCGGCTGGAGGCCCACCGTGGTCCTGGATGATGGCATCGATAAATTAATCAAGAGCATTCGATGCAAGTAA

[0561] SEQ ID NO 46: Escherichia coli NDP-4-keto-6-deoxy-glucose 4-ketoreductase (EcFCD) translated nucleotide sequence (316aa):

[0562] MDARKNGVLITGGAGFIGKALITEMVERQIPLVSFDISDKPDSLPELSEYFNWYKFSYLESSQRIKELHEIVSRHNIKTVIHLATTMFPHESKKNIDKDCLENVYANVCFFKNLYENGCEKIIFASSGGTVYGKSDTPFSEDDALLPEISYGLSKVMT ETYLRFIAKELNGKSISLRISNPYGEGQRIDGKQGVIPIFLNKISNDIPIDIIGSIESKRDYIYISDLVQAFMCSLEYEGHEDIFNIGSGESITLKKLIETIEFKLNKKAVIGFQDPIHTNANGIILDIKRAMAELGWRPTVVLDDGIDKLIKSIRCK*

[0563] SEQ ID NO 47: Nucleic acid sequence encoding the QsFSL-1 enzyme according to SEQ ID NO 48. QsFSL-1

[0564] ATGGCAGAAGCAACAGAGAGGTATGCTGTTGTGACAGGATCTAATAAAGGAATTGGATTTGGGATATGCAAGCAGCTGGCTTCTAAGGGGATTACAGTAGTGCTAACAGCTAGAGATGATAAGAGAGGTCTTGAAGCAGTTGAGAAATTGAAAGAATTTGATCTGCATGGTCATGTGCTTTTTCATCAACTTGATGTGTCTGATACAGCTAGTGTTACTAGCCTTGCAGATTTTATCAAAACCCAGTTTGGGAAACTAGATATCTTGGTAAACAATGCAGGTATAACTGGAACCACTGTAGATGCTGATGCTTTAGCATCTTCAGGCTATGGTACTGGGGGTGAACGTAAACCTATTGATTGGAATAAAATAGTGATAGAGACTTATGAATCAGTTGAAAAAGCTATCAACACCAACTATTATGGAGCCAAAAGAATGGCTGAAGCACTTATACCCCTTCTTCAAGTATCAGACTCACCAAGGATTGTTAATGCTTCCTCTCCTATGGCAAAGCTAGAGAATATTCCAAGTGGATGGGGTAAGGAAGTGCTAAGTGATGTTGATAGCCTAACAGAAGAGAAACTTGATGAGATGTTGACCCAATTATTGAAAGATTTCAAAGAGGGTTCATTAGAAACCAAAGGCTGGCCTACTCTTATGTCTTCGTATATAATCTCAAAAGCTGCTTTAAATGCCTACACAAGGATTCTTGCTAAGAAGTACCCATCTTTCTGCATCAATTGTGTAGACCCTGGTCATGTGAAGACTGACATAAATCGTCACACCGGCCACTTAAGTATTGATGAAGGTGCTGAAAGCCATGTGAGATTGGCCCTGCTGCCTGATGGTGGCCCTTCTGGACATTTCTTCTCCAGGACTGAAGAGACACCATTTTGA

[0565] SEQ ID NO 48: Oxidoreductase (QsFSL) that can enhance the activity of fucosyltransferase

[0566] MAEATERYAVVTGSNKGIGFGICKQLASKGITVVLTARDDKRGLEAVEKLKEFDLHGHVLFHQLDVSDTASVTSLADFIKTQFGKLDILVNNAGITGTTVDADALASSGYGTGGERKPIDWNKIVIETYESVEKAINTNYYGAKRMAEALIPLLQVSDSPRIVNASSPMAKLENIPSGWGKEVLSDVDSLTEEKLDEMLTQLLKDFKEGSLETKGWPTLMSSYIISKAALNAYTRILAKKYPSFCINCVDPGHVKTDINRHTGHLSIDEGAESHVRLALLPDGGPSGHFFSRTEETPF*

[0567] SEQ ID NO 49: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 50. QsFSL-2

[0568] ATGGGTTCAGATGGAAGGGATGTAGCAGAGAGGTATGCAGTGGTTACAGGTGCAAACAAAGGCATAGGCCTAGAAACCGTGCGGCAACTAGCGTCTCACGGCATTACAGTTGTGTTGACAGCTCGAGATGAGAAGAGAGGGACTGAAGCCACAAGAAAGCTCCACCAGCTGGGTTTGTCAAATTTGATTTTCCATCAGCTGGATGTTTTAGACCCTGTTAGCATTCAGTCACTGGCCAAGTTCATCCAAGACAAATTTGGCAGGCTTGATATCCTGGTTAATAATGCTGGAGCATCTGGACTTGCAGCTGATGAGAAAGCTCTGAAGGCATTAAACATAGATAATGCAGCTTGGCTCTCAGGCAAGGCCGCCAATTTAGTTCAAGGAATTGTCACACATACCTATGAGCAAGGCGAAGAATGCATAAATACAAACTATTATGGTGTCAAAAGGGTGACGGAAGCTCTCCTACCGCTGTTACAACTTTCCCCTATAGGAGCAAGGATAATAAATGTTTCCTCTTGCAGGGGTGAGCTAAAGAGGATTCCAATGAACGTAAGAAATGAACTGGGCGACATCAAAGTTCTGACTGAAGGCAGAATAGATGCAATTTTGATGAAATTTCTACACGATTTTAAGGATAATGCACTTGAGTCCAACGGATGGACATTGATGGGGCCTGCTTATAGCATTTCGAAGGCCAGTCTCAATGCCTACACTAGACTTCTTGCCAAAAAGTACCCCGAGATGCTCATTAACTGTGTTCATCCTGGTTATGTCAACACAGATATGACTTGGCATAGAGGGATACTGACGGTAGAAGAGGGTGCTAAAGGCCCAGCCATGCTAGCTCTTTTGCAAGATGGAGGACCTACAGGTTGCTATTTTGATAGTACTCAACAGGCAGAATTTTAA

[0569] SEQ ID NO 50: Oxidoreductase (QsFSL-2) capable of enhancing the activity of fucosyltransferase

[0570] MGSDGRDVAERYAVVTGANKGIGLETVRQLASHGITVVLTARDEKRGTEATRKLHQLGLSNLIFHQLDVLDPVSIQSLAKFIQDKFGRLDILVNNAGASGLAADEKALKALNIDNAAWLSGKAANLVQGIVTHTYEQGEECINTNYYGVKRVTEALLPLLQLSPIGARIINVSSCRGELKRIPMNVRNELGDIKVLTEGRIDAILMKFLHDFKDNALESNGWTLMGPAYSISKASLNAYTRLLAKKYPEMLINCVHPGYVNTDMTWHRGILTVEEGAKGPAMLALLQDGGPTGCYFDSTQQAEF*

[0571] SEQ ID NO 51: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 52. SoFSL-1

[0572] ATGGCTGAAGCATCCTCATTTCTTGCACAGAAAAGGTATGCGGTCGTGACAGGAGCAAACAAAGGACTAGGACTAGAAATATGCGGACAGCTTGCTTCACAGGGGGTGACGGTACTGCTGACATCCAGAGATGAAAAACGAGGCTTAGAAGCCATTGAGGAGCTTAAGAAATCGGGGATTAATTCGGAAAATCTTGAATATCATCAGCTGGATGTTACTAAGCCAGCTAGTTTCGCTTCTCTGGCCGATTTCATCAAGGCCAAATTTGGCAAGCTTGATATCCTGGTGAACAATGCAGGGATCAGCGGTGTTATTGTAGATTATGCAGCTTTAATGGAAGCCATTCGCCGTCGAGGGGCAGAGATCAATTACGATGGAGTGATGAAACAGACCTACGAGCTAGCAGAGGAATGCTTGCAAACAAATTACTATGGTGTGAAAAGAACCATTAATGCTCTCCTTCCGCTACTTCAGTTTTCCGATTCACCAAGGATCGTCAATGTTTCCTCCGATGTTGGCCTCCTTAAGAAAATACCCGGCGAGAGAATCAGAGAAGCCTTAGGCGACGTGGAAAAACTTACGGAAGAAAGCGTGGACGGGATTTTAGACGAGTTTCTAAGAGATTTCAAGGAAGGCAAGATCGCAGAGAAAGGTTGGCCTACGTTTAAGAGCGCCTATTCAATCTCAAAGGCGGCGCTCAATTCGTACACGAGGGTTTTAGCACGGAAATACCCGTCGATCATCATCAACTGTGTCTGCCCGGGTGTCGTCAAAACCGATATCAATCTTAAAATGGGCCACTTGACGGTTGAAGAAGGCGCGGCCAGTCCCGTGAGGTTAGCACTCATGCCCCTTGGTTCGCCTTCCGGCCTGTTCTATACTCGAAACGAAGTAACTCCATTTGAATGA

[0573] SEQ ID NO 52: Oxidoreductase (SoFSL-1) capable of enhancing the activity of fucosyltransferase

[0574] MAEASSFLAQKRYAVVTGANKGLGLEICGQLASQGVTVLLTSRDEKRGLEAIEELKKSGINSENLEYHQLDVTKPASFASLADFIKAKFGKLDILVNNAGISGVIVDYAALMEAIRRRGAEINYDGVMKQTYELAEECLQTNYYGVKRTINALLPLLQFSDSPRIVNVSSDVGLLKKIPGERIREALGDVEKLTEESVDGILDEFLRDFKEGKIAEKGWPTFKSAYSISKAALNSYTRVLARKYPSIIINCVCPGVVKTDINLKMGHLTVEEGAASPVRLALMPLGSPSGLFYTRNEVTPFE*

[0575] SEQ ID NO 53: Nucleic acid sequence encoding the SpolFSL enzyme according to SEQ ID NO 54. SpolFSL

[0576] ATGGCTGAACAATCCAACTTTCTGGCTGAAAAAAGGTATGCAGTAGTGACAGGTGCAAACAAAGGAATAGGGCTTGAAATATGCAGACAGCTTGCTTCTCAAGGTGTGATTGTACTTATCACTTCTAGAGATGGAAAGAAAGGATTAGAAGCCCTTAATGATCTCATTAAATCTGGAATTAGCTCTGATAATCTTCATTATCATCAGCTTGATGTTACTGACCCTATGAGTATTACTGCTCTTGCTGGTTTCATCAATTCCAAATTTGGCAAGCTTGATATTCTGGTGAACAATGCTGGGATAGGTGGATTTATAATTGACTACGATGCTATCAAAGCAATAGGTTTTCGCAATATCAATTATGACGAGATGATGACACAAACATATGAGCTTGCAAAAGAATGCTTGGAAACAAACTACTATGGAGTTAAGAGAACAACTGAAGCTTTGCTTCCTCAGCTGGAGTTATCGGATTCACCAAGGATCATCAATGTCTCCTCTTCTACGGGGATGTTGAAGAATATACCAAATGAGAGGATCAGAGGAGTCTTGGGTGATGCAGAGAATCTTACAGAAGAAAAAGTTGAAGCGATTTTGAATGAGTTACTGACAGATTTCAAAGATGGTTCATTCAAAGAGAAAGAATGGCCTTCTAGAATGGCAGCTTATACACTGTCAAAGGCGGCTTTGAATGCATATGCAAGAATATTGGCTAAGAAATACCCGTCAATTATCATCAGTTGTGTTTGTCCTGGTGTTACTAAGACAGATATGAACGGAAACTTGGGACAATTAACAGTTGAAGAAGGGGCCGCAAGTCCGGTGAGAGTAGCATTGATGCCTCATGGTTCACCTTCCGGTCTTTTCTATGCAAGAAGCGAAGTTTCTTCATATGAATAA

[0577] SEQ ID NO 54: Oxidoreductase (SpolFSL) capable of enhancing the activity of fucosyltransferase

[0578] MAEQSNFLAEKRYAVVTGANKGIGLEICRQLASQGVIVLITSRDGKKGLEALNDLIKSGISSDNLHYHQLDVTDPMSITALAGFINSKFGKLDILVNNAGIGGFIIDYDAIKAIGFRNINYDEMMTQTYELAKECLETNYYGVKRTTEALLPQLELSDSPRIINVSSSTGMLKNIPNERIRGVLGDAENLTEEKVEAILNELLTDFKDGSFKEKEWPSRMAAYTLSKAALNAYARILAKKYPSIIISCVCPGVTKTDMNGNLGQLTVEEGAASPVRVALMPHGSPSGLFYARSEVSSYE*

[0579] SEQ ID NO 55: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 56.

[0580]

[0581] SEQ ID NO 56: Glucosyltransferase capable of transferring a glucose residue to the C-3 position of the C-28 rhamnose residue of a QA-Tri(X / A)-F* derivative (Qs-7-GlcT)

[0582] MADRVINSYKKLHVVLFPWLAFGHMIPFLELAKLIAQKGHKISYISTPRNIRRLPKIPSHLSNNLNFIEFPLPHIPNLHENVESTNDVTHDNPIAYYLLIKALEGLQQP ITTFLETSDPDWIIHDVFPQWITATASRLRISHAFYTTSSALRTASNYSPLMSELPQDIATDFTTKSTNLLAAKVLVIRSCLELEPKEFEQCKNLCVSKTVIPLGVVPP SIQVNDNISNINDDDNDWVKIVEWLNQGKEKGSVIYVALGSEVSLSEQDLKEFALGLELSGLSFFWVFRNTGSYGLPAGFFEDRVKGRGIVWTSWAPQVSILGHESIGGFL SHGGWSSVIESLSFGIPLVVFPFGADQGINAKQLEGKNAGVEIPRSEGTGSFTRKSVADLLRLVVVEEEGKVYRDGAKELRKLFGDKDLNHKYIDNFVKYMEEHITNAAN

[0583] SEQ ID NO 57: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 58.

[0584]

[0585] SEQ ID NO 58: Rhamnosyltransferase capable of transferring a rhamnose residue to the C-3 position of D-fucose of the C-28 chain of a QA-Tri(X / A)-F* derivative (Qs-7-RhT)

[0586] MTSNNSQLHIFFLPALSPGHMIPVIEMAKLVASRGVMATIVTTAHNLAFVSRTISTYSTKIKIVTIKFPYAEVGLPEGCEIIDSDTPPDILFRVIKALRLLQEPMEQLLSSYQPDCLVAD AFSWATNSAAKFNIPRLVFHVACLFSLCASHSIELYEPQKKVSSDSETFIIPSLPGEIKLTKMQLPADLPKTGVEAEYINKMVKAVHESVENSYGFIINSFYELEKDYVDYYRNVIGRKA WHIGPLSLCHADNIEEKSQRGKESSIAENECLKWLDSRKPDSVVYVGFGSLVNFSDSQLMEIALGLEASEKQFIWVVKKSKRNEQEKEEWLPEGFEKRTVGKGLIIRGWAPQLLIMDHEAV GGFVTHCGWNSTLEGVCGGVVMATWPVSYEQIYTEKLVTDVLKIGVSVGAQTCDGIVGGIIKSEAIEKAVNRIMEGIEAEEMRSRAKAFAKKARQSVKEGGSSYSDLNSLIEELSLKSLKH

[0587] SEQ ID NO 59: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 60.

[0588]

[0589] SEQ ID NO 60: Acetyltransferase capable of transferring an acetyl group to the C-4 position of D-fucose of the C-28 chain of a QA-Tri(X / A)-F* derivative (Qs-7-AcetylT)

[0590] MKIETISTNCIKPSKPTPSHLRNIKLSDQHQHSPDVHSNFTFFYQSNQIDDAVVTVPSAAIDVAPATDAAIDVAAATDTAIDGAATNFSVQSKILHNCLATTLTSFYPIAGRFQNGDTIICN DEGAFFIEAKTDINMSNFLGHPDLLTVIREHLVPDATNPDYNGSILLLKFTLFGCGSTAITISMSHKIADLVTFITLLNCWTALARAGGGGGIGGGSDGFIPPDLNFLGQIVPDSDPSPKSAT PEFFRNKKFVTKRFVFSASKIKEVKDKVMKEIRKQEDDIFPSRVDVVLALIWRSTLSSLGSSGKFKPAIFMQAANLRTRTDPPLPETSIGNLVILFPLVVEKETDIELHELVNKLLDAKAWV NKLKKKFQGYDGGNDPLQVVEAIRCEALKEMGNVWKKSKDFSMYISSSFCNFLMNEVDFGWGKPVWVTNTPRTIMANTIYLLDTKEVGGVDALVQFEEEEITKLELNQELLQFATVNPIPIVI

[0591] SEQ ID NO 61: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 62.

[0592]

[0593] SEQ ID NO 62: Acetyltransferase (SOAP10) capable of transferring an acetyl group to the C-4 position of a D-fucose residue on the QA-Tri(X / A)-F* scaffold

[0594] MGEVNHEEVEIEIISIETIKPSSLLPPKTPPKTITLSHLDQAAPLYYYPLLLYYTNTTTTTPTSQIRVDITSTLKTSLSKTLDKFHPIAGRCVDDSTICCNHQGIPFIETKVDSNILDVMNSPEKMKL LIKFLPHAEFQDVTRPVSDLNHLAFQVNVFRCGGVIIGSYVLHKLLDGISLGTFFKNWSTIANDERVKDDDLVQPDFEATIKAFPPRTATPMLPRNQQLPKAAEKPNNNPVKVLVTKSFVFFDIVSLKK MMFMAKSELVPKPTKFETVTGFIWEQTLSTLRNSGVEVEHTSLIIPVNIRPRMSPPLPRGSMGNLKNAKAQANTSSSNGLQDLVKEIHSSLSQTTQKINTPPPPPPPPPTTTATTIHSSLSQTTQKIN TPPPTTTTIHSSLSQTTQKINTTTTTTAEVILTKRKVDNPVTQNREGNYLFTSWCKIGLDEADFGFGKPVWVIPNDGRPPKVRNMIFLTDYRHPETGVEGIAAWITLEEKQMQCLKSNPEFLAFATPN

[0595] SEQ ID NO 63: Nucleic acid sequence encoding the enzyme according to SEQ ID NO 64.

[0596] ATGATGGAGGTACATACCACATCGGAAAATTGCATTAAGCCCTCACAACCCACTCCTTCCCACCTTCAAAATTTGAAACTCTCTAATCATCATAGCCAAGCACCCGATATCCGTACCAACCTCACCTTCTTCTTCTCCTCTAATTTTAACAATCCAGTCCAGCCTGGTGACCATGACGCTACTACCAATTTTACACTCCAATCCAAACTTGTTCAGAATTCATTGGCTACAACTCTCACAATTCTCTACCCTTTTGCTGGTCGATTCCGAAACGACGACACCATCATTTGCAAAGACGATGGCGCCTTCTTTATTGAAGCAAAAACCGACACCAAACTTTCTGACTTTCTTGCCCAGCCGGACTTGCCTCTAGCTATAATGGACAAATTAGTCCCCGTAGCTACCGACGCCAAGTATAATGGTTCTCTGCTAATCCTGAAATTTACTTTGTTTGGCTGCGGTGGCTCAGCCGTAACCATCTCAATAACTCACAAGATTTCCGATCTAGCAACCATTTTGACACTGCTTAATTG CTGGACTGCTTTATCTCGTGGTGGTGATGGTGGTGGTTCCAGTCCGTTTATTCAGCCTGATTTGAATTTCATTGGCCGCCCTGTTCCAAGTACGAGTGAGGTTCCTCCTCCATCTTCTGGAAAG AATTTTATTCCTCCAAATAGCAAGTACGTTACCAAAAGGTTCATATTCAGTGCAGCAAAGATTAAAGAACTGAAAGCCAGAGTCATCAACAAGATTCGTAAAGAAGAAGACAATGTTTTCCCTTCCCGTGTGGATGTAGTGCTGGCACTCATTTGGAAATGCGCTTTGGCTTCTGTTAATTCTGGTTCCAGATCTGGAAATGCACAAACATTTAGGCCGTCGGTAATGATGCAAGCCGTGAACCTCAG AAACCGTACAGATCCACCATTACCGGGAATCTTCGATTGGGAACTTGGCAATACTATTACCGGTGTGGGTGGAGAAAGAGGAAGACACGGAATTACATGAACTTGTTAGCAGATTGTTAACAGTGAAAGTGCGTGCTAACAGATTGAAGAAGAAATACCAAGGGTTATGAAGATCCAGAGCAAGTTATTATTTCAATGGAATCTGATTCAGTGAAGGAAATAATAGAGGTTCGGAAGAAATTGAAGGATTTCAGTACTTATGTTGCGGCGAGTGTGGTGAATGCTCCATTGTATGACGTTGATTTTGGGTGGGGAAAACCAGCATGGGTGACCAGCACGCCCAACACAGTAATGGCGAATTCTATATATTTGTTGGATACCAAAGACGCCGGTGGGATTGAAGTTTTGATGAATATGTTGAAAGAGGAAGACATGATTGTCTTTGAAAGCAATCAGGAGTTGCTTCAGTCTGCCATGGTTAATCCCACTATCATTTGA

[0597] SEQ ID NO 64: Acetyltransferase (DMOT9) capable of transferring an acetyl group to the C-4 position of a D-fucose residue on the QA-Tri(X / A)-F* scaffold

[0598] MMEVHTTSENCIKPSQPTPSHLQNLKLSNHHSQAPDIRTNLTFFFSSNFNNPVQPGDHDATTNFTLQSKLVQNSLATTLTILYPFAGRFRNDDTIICKDDGAFFIEAKTDTKLSDF LAQPDLPLAIMDKLVPVATDAKYNGSLLILKFTLFGCGGSAVTISITHKISDLATILLTLLNCWTALSRGGDGGGSSPFIQPDLNFIGRPPVPSTSEVPPPSSGKNFIPPNSKYVTKRF IFSAAKIKELKARVINKIRKEEDNVFPSRVDVVLALIWKCALASVNSGSRSGNAQTFRPSVMMQAVNLRNRTDPPLPESSIGNLAILLPVWVEKEEDTELHELVSRLLTVKVRANR LKKKYQGYEDPEQVIISMESDSVKEIIEVRKKLKDFSTYVAASVVNAPLYDVDFGWGKPAWVTSTPNTVMANSIYLLDTKDAGGIEVLMNMLKEEDMIVFESNQELLQSAMVNPTII

Claims

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

2. The method according to claim 1, wherein: a) QA-Tri(X / R)-F* is first combined with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56, to form QA-Tri(X / R)-F*-G; Then b) combining QA-Tri(X / R)-F*-G with one or more enzymes selected from the group consisting of: QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; SOAP10 having an amino acid sequence of SEQ ID NO 62 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or DMOT9 having an amino acid sequence of SEQ ID NO 64 or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64 to form QA-Tri(X / R)-F*-G-Ac; and then c) combining QA-Tri(X / R)-F*-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 having at least 70% sequence identity with SEQ ID NO 58 to form QA-Tri(X / R)-F*-GR-Ac.

3. The method of claim 1, wherein in steps a), b) and c), F* is FRX.

4. The method according to claim 1, wherein: a) first combining QA-Tri(X / R)-F* with one or more enzymes selected from the following to form QA-Tri(X / R)-F*-Ac: 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 with 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 with 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 with SEQ ID NO 64; and then b) combining QA-Tri(X / R)-F*-Ac with an enzyme QS-7-RhaT having an amino acid sequence of SEQ ID NO 58 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 58 to form QA-Tri(X / R)-F*-R-Ac; Then c) combining QA-Tri(X / R)-F*-R-Ac with 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 with SEQ ID NO 56 to form QA-Tri(X / R)-F*-GR-Ac.

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

6. The method according to claim 1, wherein: i. firstly combining QA-Tri(X / R)-F* with one or more enzymes selected from the following to form QA-Tri(X / R)-F*-Ac: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64 or an enzyme having an amino acid sequence having at least 25% sequence identity with SEQ ID NO 64; and then ii. combining QA-Tri(X / R)-F*-Ac with an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56 or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56 to form QA-Tri(X / R)-F*-G-Ac; Then iii. QA-Tri(X / R)-F*-G-Ac is combined with 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 with SEQ ID NO 58 to form QA-Tri(X / R)-F*-GR-Ac.

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

8. The method of any one of claims 1, 2, 4 or 6, wherein Tri(X / R) is TriX and F* is FRXA.

9. A method for producing biosynthetic QA-Tri(X / R)-F*-GR-Ac in a host, the method comprising the steps of: a) expressing genes required for the biosynthesis of QA-TriR-F* and / or QA-TriX-F*, and b) introducing into the host a polynucleotide encoding: i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56; ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an 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 with SEQ ID NO 58.

10. A method for producing biosynthetic QA-TriX-F*-GR-Ac in a host, the method comprising the steps of: a) express the genes required for the biosynthesis of QA-TriX-F*, and b) introducing into the host a polynucleotide encoding: i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56; ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an amino acid sequence of SEQ ID NO 64, or an enzyme having an amino acid sequence having at least 25% sequence identity with 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 with SEQ ID NO 58. The method of claim 10 , wherein F* is FRXA.

12. A method for producing biosynthetic QA-TriR-F*-GR-Ac in a host, the method comprising the steps of: a) express the genes required for the biosynthesis of QA-TriR-F*, and b) introducing into the host a polynucleotide encoding: i. an enzyme QS-7-GlcT having an amino acid sequence of SEQ ID NO 56, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56; ii. one or more enzymes selected from the group consisting of: an enzyme QS-7-AcetylT having an amino acid sequence of SEQ ID NO 60, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60; an enzyme SOAP10 having an amino acid sequence of SEQ ID NO 62, or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 62; or an enzyme DMOT9 having an 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 with SEQ ID NO 58.

13. The method according to any one of claims 9 to 12, wherein: The amino acid sequence SEQ ID NO 60 is encoded by the polynucleotide sequence SEQ ID NO 59; The amino acid sequence SEQ ID NO 58 is encoded by the polynucleotide sequence SEQ ID NO 57; The amino acid sequence SEQ ID NO 56 is encoded by the polynucleotide sequence SEQ ID NO 55; The amino acid sequence SEQ ID NO 62 is encoded by the polynucleotide sequence SEQ ID NO 61, and the amino acid sequence SEQ ID NO 64 is encoded by the polynucleotide sequence SEQ ID NO 63.

14. A glucosyltransferase having the amino acid sequence of SEQ ID NO 56 (QS-7-GlcT), or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 56.

15. A rhamnosyltransferase having the amino acid sequence of SEQ ID NO 58 (QS-7-RhaT), or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 58.

16. An acetyltransferase having the amino acid sequence of SEQ ID NO 60 (QS-7-AcetylT), or an enzyme having an amino acid sequence having at least 70% sequence identity with SEQ ID NO 60.

17. A polynucleotide encoding any enzyme according to any one of claims 14 to 16. A vector comprising the polynucleotide according to claim 17 .

19. A host cell comprising the polynucleotide according to claim 17.

20. A host cell transformed with the vector according to claim 18.

21. A host cell according to claim 19 or claim 20, wherein the host cell is a plant cell or a microbial cell.

22. A plant or microbial biological system comprising the host cell according to any one of claims 19 to 21.

23. The biological system of claim 22, wherein the biological system is yeast or Nicotiana benthamiana.

24. The method according to any one of claims 1 to 13, wherein the method further comprises the step of isolating the QA-Tri(X / R)-F*-GR-Ac derivative.

25. A QA-Tri(X / R)-F*-GR-Ac derivative obtainable by the process of claim 24.

26. The derivative according to claim 25, wherein the derivative is 3--O-{β-D-xylopyranosyl-(1->3)-[β-D-galactopyranosyl-(1->2)]-β-D-pyranoglucuronic acid}-28-O-{β-D-apiofuranosyl-(1->3)-β-D-xylopyranosyl-(1->4)-α-L-rhamnopyranosyl-(1->2)-β-D-fucopyranosyl ester}-saponic acid, which is acetylated at the C-4 position of the D-fucose of the core C-28 chain, and the rhamnose moiety is attached to the C-3 position of the D-fucose of the C-28 chain, and the glucose moiety is attached to the C-3 position of the core C-28 rhamnose moiety (QA-TriX-FRXA-GR-Ac).

27. Use of the QA-Tri(X / R)-F*-GR-Ac derivative according to claim 25 or 26 as an adjuvant.

28. The use according to claim 27, wherein the adjuvant is a liposome or an immunostimulatory complex (ISCOM) formulation.

29. The use according to claim 28, wherein the ISCOM formulation comprises a first ISCOM matrix comprising the QA-Tri(X / R)-F*-GR-Ac derivative according to claim 25 or claim 24, and a second ISCOM matrix comprising QS-21.

30. The use according to any one of claims 27 to 29, wherein the adjuvant further comprises a TLR4 agonist.

31. The use according to claim 30, wherein the TLR4 agonist is 3D-MPL.

32. An adjuvant composition comprising the QA-Tri(X / R)-F*-GR-Ac derivative according to claim 25 or claim 26.

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