Saponin production in yeast

Abstract

The present invention relates to methods for the biosynthetic production of QS-21, its precursors and derivatives, and related embodiments.

Description

[Technical field]

[0001] The present invention relates to the biosynthetic production of QS-21, its precursors and derivatives, and unnatural sugars in yeast, and related aspects. [Background technology]

[0002] QS-21 is a natural saponin extract obtained from the bark of the Chilean soap tree, also known as Quillaja saponaria. The QS-21 extract was first identified as a fraction (peak 21) purified by RP-HPLC purification from a crude extract of Quillaja Saponaria Molina bark (Kensil et al., 1991). This crude bark extract has been reported to contain various types of saponins. The QS-21 extract or QS-21 fraction contains several different saponin molecules. The QS-21 fraction has been reported to contain two major isomeric molecular components (Ragupathi et al., 2011), which are shown in Figure 1. Each of these isomers contains a central triterpene core or aglycone (quilacic acid), a branched trisaccharide attached to an oxygen functional group at C3 of the triterpene, and a linear tetrasaccharide attached to a carboxylate group at C28 of the triterpene. The fourth component in the saponin structure is a glycosylated C18 pseudodimeric acyl chain attached to a fucose residue of the linear tetrasaccharide, which is terminally modified with an arabinofuranose residue via a hydrolytically labile ester bond. These isomeric components differ in the composition of the terminal sugar residues of the tetrasaccharide, with the major isomer incorporating an apiose sugar (65%) ("QS-21-Api") and the minor isomer incorporating a xylose sugar (35%) ("QS-21-Xyl") (see R2 in Figure 1).

[0003] Quillaja saponins, including QS-21, have long been known to have potent immunostimulatory properties that can enhance antibody production and specific T cell responses. These properties have led to the development of Quillaja saponin-based adjuvants for vaccines. Notably, the AS01 adjuvant is a liposomal formulation containing QS-21 and 3-O-desacyl-4'-monophosphoryl lipid A (3D-MPL) (Garcon, 2011; Didierlaurent, 2017), which is currently used in the development of the varicella zoster vaccine (Shingrix TM ) and a vaccine for malaria (Mosquirix TM It has been licensed as a vaccine against diseases such as polio.

[0004] While a number of vaccines containing QS-21 are becoming available, the demand for its supply is expected to increase significantly over the next few years. Thus, there is a need to provide a method for producing QS-21 that is independent of natural resources, for example, by biosynthesis in yeast. Advantages of such a method include, for example: (i) It eliminates the need for complex purification schemes designed to separate saponins from complex mixtures containing multiple types of saponins (e.g., from crude bark extracts). (ii) It is possible to produce various types of saponins (e.g., QS-21-Api and QS-21-Xyl) that are difficult to separate from crude bark extracts. (iii) It is possible to produce any desired saponin (including precursors that cannot be purified from crude bark extracts).

[0005] Biosynthetic production of QS-21 precursors has been reported in Nicotiana benthamiana (e.g., WO19 / 122259, WO20 / 260475 and WO 22 / 136563). Trace amounts of quillic acid have also been reported to be produced in yeast (WO20 / 263524). The present invention is the first to report successful production of QS-21 and its glycosylated precursors and glycosylated derivatives in yeast. Summary of the Invention [Means for solving the problem]

[0006] In a first aspect of the present invention, there is provided a method for producing quillaric acid (QA) in yeast, comprising the steps of: In yeast engineered to produce β-amyrin, (i) cytochrome P450 C16 oxidase, which oxidizes the carbon at the C16 position of β-amyrin to generate a hydroxyl group; (ii) cytochrome P450 C23 oxidase, which oxidizes the carbon at C23 of β-amyrin to generate an aldehyde group; (iii) cytochrome P450 C28 oxidase, which oxidizes the carbon at C28 of β-amyrin to generate a carboxyl group; and / or (iv) Cytochrome P450 reductase (CPR) acting as a redox partner overexpressing a heterologous gene encoding the C16 oxidase, the C23 oxidase, the C28 oxidase and the CPR are derived from a plant; According to the above method, yeast recombinantly adapted to produce QA are provided.

[0007] In a second aspect, there is provided a method for producing UDP-glucuronic acid (UDP-GlcA) in yeast, comprising the steps of: overexpressing a heterologous gene encoding UDP-glucose dehydrogenase (UGD) which converts UDP-glucose (UDC) to UDP-GlcA; According to the methods described above, yeast cells engineered to produce UDP-GlcA are provided.

[0008] In a third aspect, there is provided a method for producing UDP-rhamnose (UDP-Rha) in yeast, comprising the steps of: overexpressing a heterologous gene encoding UDP-rhamnose synthase (RhaT), which converts UDP-Glc to UDP-Rha; According to the above method, a yeast recombinantly produced to produce UDP-Rha is provided.

[0009] In a fourth aspect, there is provided a method for producing UDP-xylose (UDP-Xyl) in yeast, comprising the steps of: (i) UDP-glucose dehydrogenase (UGD), which converts UDP-Glc to UDP-GlcA; and / or (ii) UDP-xylose synthase (UXS), which converts UDP-GlcA to UDP-xylose. and overexpressing a heterologous gene encoding According to the above method, a yeast recombinantly capable of producing UDP-Xyl is provided.

[0010] In a fifth aspect, there is provided a method for producing a C3-glycosylated QA derivative in yeast, comprising the steps of: In yeast engineered to produce QA and UDP-GlcA, (i) UDP-GlcA transferase (GlcAT), which transfers UDP-GlcA to attach a GlcA residue to the C3 position of QA, thereby forming a C3-glycosylated QA derivative. and overexpressing a heterologous gene encoding According to the above method, yeast recombinantly engineered to produce C3-glycosylated QA derivatives are provided.

[0011] In a sixth aspect, there is provided a method for producing UDP-Fuc in yeast, comprising the steps of: (i) UDP-glucose-4,6-dehydratase (UG46DH), which converts UDP-Glc to UDP-4-keto-6-deoxyglucose, and / or (ii) 4-ketoreductase that converts UDP-4-keto-6-deoxyglucose to UDP-Fuc and overexpressing a heterologous gene encoding According to the above method, a yeast recombinantly produced UDP-Fuc is provided.

[0012] In a seventh aspect, there is provided a method for producing a C28-glycosylated QA derivative in yeast, comprising the steps of: In yeast engineered to produce C3-glycosylated QA derivatives, (i) UDP-fucose transferase (FucT), which transfers UDP-Fuc to attach a Fuc residue to the C28 position of a C3-glycosylated QA derivative, thereby forming a C28-glycosylated QA derivative; and overexpressing a heterologous gene encoding According to the above method, yeast recombinantly engineered to produce a C28-glycosylated QA derivative are provided.

[0013] In an eighth embodiment, there is provided a method for producing (S)-2-methylbutyryl-CoA (2MB-CoA) in yeast, comprising: In the presence of exogenously added 2-methylbutyric acid (2MB), Carboxyl coenzyme A (CoA) ligase (CCL) converts 2-methylbutyrate (2MB) to 2MB-CoA and overexpressing a heterologous gene encoding According to the above method, a yeast recombinantly engineered to produce 2MB-CoA is provided.

[0014] In a ninth aspect, there is provided a method for producing UDP-arabinofuranose (UDP-Araf) in yeast, comprising: In yeast engineered to produce UDP-Xyl, (i) a UDP-Xyl epimerase (UXE) that converts UDP-Xyl to UDP-arabinopyranose (UDP-Arap); and / or (ii) UDP-arabinose mutase (UAM), which converts UDP-Arap to UDP-arabinofuranose (UDP-Araf) and overexpressing a heterologous gene encoding According to the above method, a yeast recombinantly produced by the method is provided.

[0015] In a tenth aspect, there is provided a method for producing an acylated glycosylated QA derivative in yeast, comprising the steps of: In yeast engineered to produce glycosylated QA derivatives, (i) carboxyl coenzyme A (CoA) ligase (CCL), which converts 2-methylbutyrate to (S)-2-methylbutyryl CoA (2MB-CoA); (ii) a chalcone synthase-like type III polyketide synthase (PKS) that condenses malonyl-CoA with 2MB-CoA to form C9-keto-CoA; (iii) a ketoreductase (KR) that converts C9-keto-CoA to C9-CoA; and / or (iv) an acyltransferase that transfers and attaches a first C9-CoA unit to the glycosylated QA derivative to form an acylated glycosylated QA derivative; overexpressing a heterologous gene encoding 2-methylbutyric acid may be added exogenously. According to the above method, yeast recombinantly engineered to produce acylated glycosylated QA derivatives are provided.

[0016] In an eleventh aspect, there is provided a QA derivative obtainable by the method according to any one of the first to tenth aspects of the present invention.

[0017] In a twelfth aspect, there is provided the use of a QA derivative according to the eleventh aspect of the invention as an adjuvant.

[0018] In a thirteenth aspect, there is provided an isolated enzyme or protein for use in the methods according to the first to tenth aspects of the present invention. [Brief description of the drawings]

[0019] [Figure 1] The structures of the two major isomeric components present in the QS-21 fraction, conventionally purified from crude bark extracts obtained from Quillaja saponaria Molina trees, are shown. The core skeleton is formed from the triterpene quillaric acid (QA). The C3 position of QA is characterized by a branched trisaccharide consisting of a β-D-glucuronic acid (β-D-GlcA) residue, a β-D-galactose (β-D-Gal) residue, and a β-D-xylose (β-D-Xyl) residue at the R1 position. The C28 position of QA is characterized by a linear tetrasaccharide consisting of a β-D-fucose (β-D-Fuc) residue, an α-L-rhamnose (α-L-Rha) residue, a β-D-xylose residue, and a terminal β-D-apiose (β-D-Api) residue or a terminal β-D-xylose residue at the R2 position. Furthermore, the β-D-fucose residue features an 18-carbon pseudodimeric acyl chain terminally modified with an α-L-arabinofuranose (α-L-Araf) residue. The carbon numbers of QA (C3, C16, C23, and C28) are illustrated. Substitution of an α-L-rhamnose (α-L-Rha) residue at the R1 position represents a rhamnose-derived species of QS-21, which is present in trace amounts in the QS-21 fraction conventionally purified from crude bark extracts obtained from Quillaja trees.

[0020] [Diagram 2]Biosynthetic pathways for the de novo production of QS-21 in yeast. Figure 2A shows the biosynthesis of the nucleotide sugars required for the C3 branched trisaccharide and C28 linear tetrasaccharide, and the C9-CoA building blocks of the 18-carbon pseudodimeric acyl chain from the mevalonate pathway. "UGE" is UDP-glucose 4-epimerase, "UGD" is UDP-glucose dehydrogenase, "RHM" is rhamnose synthase, "UXS" is UDP-xylose synthase, "AXS" is UDP-apiose / UDP-xylose synthase, "UXE" is UDP-xyl epimerase, and "UAM" is UDP-arabinose mutase. "LovF-TE" is a polyketide synthase (PKS) (or sometimes called "megasynthase"). "ACP" is an acyl carrier protein. "TE" is a thioesterase. "CCL" is carboxyl coenzyme A ligase, "PKS" is polyketide synthase, and "KR" is ketoreductase. Figure 2B shows the biosynthesis of quilacic acid by sequential oxidation of β-amyrin. "BAS" is β-amyrin synthase. Figure 2C shows the biosynthesis of the branched trisaccharide at the C3 position of QA. "GlcAT" is UDP-glucuronate transferase. "GalT" is UDP-galactose transferase. "XylT" is UDP-xylose transferase. "RhaT" is UDP-rhamnose transferase. Figure 2D shows the biosynthesis of the linear tetrasaccharide at the C28 position of QA. "FucT" is UDP-fucose transferase. FIG. 2E shows the addition of an 18-carbon pseudodimeric acyl chain to the fucose residue of the linear tetrasaccharide at C28 of QA, followed by the addition of an arabinofuranose to the terminus of this acyl chain.

[0021] [Diagram 3]Screening of β-amyrin synthases (BAS) from various types of plants. The abundance of β-amyrin in yeast recombinant with genes encoding a BAS from Artemisia annua (Aa) ("AaBAS"), a BAS from Arabidopsis thaliana (At) ("AtBAS"), a BAS from Glycyrrhiza glabra (Gg) ("GgBAS") and / or a BAS from Gypsophila vaccaria (Gv) ("GvBAS") was measured by GC-MS 1, 2 and 3 days after induction of gene expression.

[0022] [Figure 4] The production of QA precursors (gypsogenin, oleanolic acid and hederagenin) and QA in various yeast strains engineered with various combinations (as shown) of the enzymes and proteins listed in Table 3 is shown.

[0023] [Diagram 5] We compared the subcellular localization of cytochrome P450 C28 oxidase from Quillaja japonica (QsC28) (Fig. 5A), cytochrome P450 C16 oxidase from Quillaja japonica (QsC28) (Fig. 5B), and an oxidase derived from a fusion of QsC28 to the N-terminus of QsC16 (QSC28C16) (Fig. 5C). These oxidases were tagged with fluorescent proteins (GFP or mCherry (as shown)) at their C-termini.

[0024] [Figure 6]Panel A shows the relative expression of β-amyrin synthase (SvBAS) mRNA in leaves treated with 0 μM, 50 μM, or 100 μM methyl jasmonate (MeJa) for 72 h. Panel B shows the fold change (compared to 0 μM) of β-amyrin synthase in flowers treated with 50 μM or 100 μM MeJa at 24 and 72 h. Panel C shows a neighbor-joining phylogenetic tree showing cytochrome P450 (CYP) candidates identified from the Saponaria baccaria transcriptome along with cytochrome P450 (CYP) from other plants that act on triterpenoids. Gene names labeled with an asterisk indicate genes in Saponaria baccaria. Boxed gene names indicate CYPs co-expressed with β-amyrin synthase.

[0025] [Figure 7] (Panel A) LC-MS extracted ion chromatograms (EICs) of QA precursors ("oleanolic acid" and "echinocystic acid") detected in Nicotiana benthamiana plants transiently co-expressing various combinations (as shown) of β-amyrin synthase from Saponaria vacccaria ("SvBAS"), CYP C28 oxidase from Saponaria vacccaria ("SvC28"), and CYP C16 oxidase from Saponaria vacccaria ("SvC16"), respectively. (Panel B) LC-MS extracted ion chromatograms (EICs) of the QA precursor (gypsogenin) detected in N. benthamiana plants transiently co-expressing various combinations (as shown) of β-amyrin synthase from Quillaja japonica ("QsBAS"), CYP C28 oxidase from Quillaja japonica ("QsC28"), CYP C23 oxidase from Saponaria vaccaria ("SvC23-1"), and CYP C23 oxidase from Saponaria vaccaria ("SvC23-2") (Panel C) LC-MS extracted ion chromatograms of the QA precursor (gypsogenic acid) detected in N. benthamiana plants transiently co-expressing the same enzyme combinations as in Panel B.

[0026] [Figure 8]LC-MS extracted ion chromatograms (EICs) of QA precursors (oleanolic acid "OA", hederagenin / echinocystic acid "Hed / EA", gypsogenin "Gyp", and echinocystic acid "EA") and QAs detected in yeast co-expressing β-amyrin synthase ("GvBAS"), CYP C16 oxidase ("SvC16"), CYP C28 oxidase ("QsC28"), CYP reductase ("AtATR1"), and CYP C23 oxidase from Saponaria vacccaria ("SvC23-1"). The dashed lines indicate that the peaks obtained in the EICs of QAs (from the "yeast sample") match those obtained in the EICs of the QA standards ("commercial QA standards"). The numbers in parentheses indicate the m / z (mass-to-charge) values.

[0027] [Figure 9] LC-MS extracted ion chromatograms (EICs) of QA precursors (oleanolic acid "OA", hederagenin / echinocystic acid "Hed / EA", gypsogenin "Gyp", and echinocystic acid "EA") and QAs detected in yeast co-expressing β-amyrin synthase ("GvBAS"), CYP C16 oxidase ("SvC16"), CYP C28 oxidase ("QsC28"), CYP reductase ("AtATR1"), and CYP C23 oxidase from Saponaria vaccaria ("Sv-C23-2"). The dashed lines indicate that the peaks obtained in the EICs of QA (from the "yeast sample") match those obtained in the EICs of the QA standard ("commercial QA standard"). Numbers in parentheses indicate m / z (mass-to-charge ratio) values.

[0028] [Figure 10] Expression profiles of AtMSBP homologs in leaves and flowers of Saponaria vacccaria (as indicated on the graph). The average expression levels of the various homologs in leaves and flowers are shown as trimmed mean of M-values ​​(TMM).

[0029] [Figure 11]FIG. 1 shows a comparison of the production of QA precursors (gypsogenin, oleanolic acid, hederagenin and erythrodiol) and QA in the presence or absence (YL-4) of MSBP proteins (as indicated on the graph) from various plants as measured by LC-MS.

[0030] [Figure 12] Biosynthetic pathways for the de novo production of nucleotide sugars in yeast via nucleotide sugar interconverting enzymes. Unnatural sugars in yeast are circled. Heterologous enzymes required for the synthesis of these unnatural sugars are underlined. "Rham synthase" is rhamnose synthase, "UGE" is UDP-glucose 4-epimerase, "UGD" is UDP-glucose dehydrogenase, "UXS" is UDP-xylose synthase, "AXS" is UDP-apiose / UDP-xylose synthase, "UXE" is UDP-xyl epimerase, "UAM" is UDP-arabinose mutase, "UG46DH" is UDP-glucose-4,6-dehydratase, and "UG46DGR" is UDP-4-keto-6-deoxyglucose reductase.

[0031] [Figure 13] Panel A shows the results of comparing the production of UDP-glucose (UDP-Glc), UDP-glucuronic acid (UDP-GlcA), and UDP-xylose (UDP-Xyl) between two yeast strains (SC-1 and SC-4) overexpressing AtUGD (UDP-glucose dehydrogenase). Panel B shows the results of comparing the production of UDP-Xyl in two yeast strains (SC-4 and SC-16) overexpressing AtUGD with UDP-xylose synthase from Arabidopsis thaliana (AtUXS) or UDP-apiose / UDP-xylose synthase from Quillaja japonica (QsAXS) 24 and 48 hours after induction of gene expression.

[0032] [Figure 14]Figure 1 shows the results of comparing the production of UDP-rhamnose (UDP-Rha), UDP-xylose (UDP-Xyl) and UDP-fucose (UDP-Fuc) in five yeast strains (SC-17, SC-19, SC-20, SC-22 and SC-23) (shown on the graph) overexpressing different combinations of enzymes (shown on the graph) from different plants. Panel A shows the results on the graph plotted against yeast strain and Panel B shows the results on the graph plotted against UDP-sugar.

[0033] [Figure 15] The production of glucuronidated QA precursors ("oleanolic acid-GlcA", "gypsogenin-GlcA" and "hederagenin-GlcA") and glucuronidated QA ("QA-C3-GlcA") is measured by LC-MS in yeast (YL-11) that produces QA and has been engineered to overexpress UDP-glucuronosyltransferase from Quillaja japonica ("QsCslG1") and UDP-glucose dehydrogenase from Arabidopsis thaliana ("AtUGD"). The left panel shows QA precursors and (non-glycosylated) QA. The right panel shows glucuronidated QA precursors and glucuronidated QA ("QA-C3-GlcA").

[0034] [Figure 16] Figure 1 shows the production of glucuronidated QA precursors ("oleanolic acid-GlcA", "gypsogenin-GlcA", and "hederagenin-GlcA") and glucuronidated QA ("QA-GlcA") measured by LC-MS in yeast (YL-12) that produces QA and has been engineered to overexpress UDP-glucose dehydrogenase ("AtUGD") and UDP-glucuronosyltransferase ("QsCslG2"). The left panel shows QA precursors and (non-glycosylated) QA. The right panel shows glucuronidated QA precursor and glucuronidated QA ("QA-C3-GlcA").

[0035] [Figure 17]The results of comparing the substrate specificity of QsCslG1 and QsCslG2 are shown in Figure 16. The data shown in Figure 16 are a graph of the quantification of the production of QA precursors ("OA", "Her" and "Gyp"), QA, glucuronidated QA precursors ("GlcA-OA", "GlcA-Her" and "GlcA-Gyp") and glucuronidated QA ("QA-C3-GlcA") (as shown) from YL-11 (overexpressing QsCslG1) and YL-12 (overexpressing QsCslG2).

[0036] [Figure 18] LC-MS extracted ion chromatograms (EICs) of QA and QA-C3-GlcA detected in an in vitro enzyme assay are shown. A microsomal preparation from yeast in which UDP-glucuronosyltransferase from Saponaria vacccaria ("SvCslG") was overexpressed from a plasmid was added directly to the reaction buffer along with QA and UDP-GlcA (both commercially available).

[0037] [Figure 19] Figure 1 shows LC-MS extracted ion chromatograms (EICs) of QA and C3-glycosylated QA derivatives ("QA-C3-GlcA" and "QA-C3-GlcA-Gal") detected in yeast (YL-13) engineered to produce QA-C3-GlcA and overexpress galactose transferase from Quillaja quinolones ("QsGalT"). Peaks corresponding to QA and QA-C3-GlcA-Gal are labeled.

[0038] [Figure 20]LC-MS extracted ion chromatograms (EICs) of C3-glycosylated QA derivatives ("QA-C3-GlcA-Gal" and "QA-C3-GlcA") detected in N. benthamiana plants transiently co-expressing Saponaria vacccaria UDP-glucuronosyltransferase ("SvCslG") and Saponaria vacccaria UDP-galactosetransferase ("SvGal") (or N. benthamiana plants transiently expressing SvCslG alone) when QA (commercially available) was infiltrated into the plants (as indicated in the graph). Peaks corresponding to QA-C3-Gal and QA-C3-GlcA-Gal are labeled.

[0039] [Figure 21] LC-MS extracted ion chromatograms (EICs) of QA and C3-glycosylated QA derivatives ("QA-C3-GlcA", "QA-C3-GlcA-Gal" and "QA-C3-GlcA-Gal-Rha") detected in yeast (YL-14) engineered to produce QA-C3-GlcA-Gal and overexpress UDP-rhamnose synthase from Arabidopsis ("AtRHM2") and UDP-rhamnose transferase from Quillaja japonica ("QsC3RhaT"). Peaks corresponding to QA and QA-C3-GlcA-Gal-Rha are labeled.

[0040] [Figure 22] LC-MS extracted ion chromatograms (EICs) of QA and C3-glycosylated QA derivatives ("QA-C3-GlcA", "QA-C3-GlcA-Gal" and "QA-C3-GlcA-Gal-Xyl") detected in yeast (YL-15) engineered to produce QA-C3-GlcA-Gal and overexpress UDP-xylose synthase from Arabidopsis ("AtUXS") and UDP-xylose transferase from Quillaja ("QsC3XylT"). Peaks corresponding to QA and QA-C3-GlcA-Gal-Xyl are labeled.

[0041] [Figure 23] Comparison of the production of QA-C3-GlcA-Gal-Xyl in seven yeast strains (YL-15, YL-16, YL-17, YL-18, YL-19, YL-20, and YL-21) that produce QA-GlcA-Gal and have been engineered to overexpress one of the various UDP-glucose dehydrogenases ("UGD variants"; as shown in the graph). "Syn" stands for Synechococcus sp., "Hs" stands for Homo sapiens, "Patl" stands for Paramoeba atlantica (Patl), "Bcyt" stands for Bacillus cytotoxicus, "Myxfulv" stands for Corallococcus macrosporus, and "Pfu" stands for Pyrococcus furiosus.

[0042] [Figure 24] Figure 1 shows a comparison of the production of QA and QA-C3-GlcA-Gal-Xyl ("QA-C3-GGX") in two yeast strains (YL-15 and YL-22) (as indicated on the graph). Compared to YL-15, YL-22 additionally overexpresses glucuronidase from Arabidopsis thaliana and UDP-glucuronate pyrophosphorylase from Arabidopsis thaliana ("AtUSP").

[0043] [Diagram 25]Figure 1 shows a comparison of the production of QA and QA-C3-GlcA-Gal-Xyl ("QA-C3-GGX") in different yeast strains (YL-15 and YL-23) under various conditions (as indicated on the graph). Compared to YL-15, YL-23 additionally overexpresses glucuronidase from Arabidopsis thaliana, UDP-glucuronate pyrophosphorylase from Arabidopsis thaliana ("AtUSP"), and myo-inositol oxygenase from Thermothelomyces thermophilus (Tt) ("TtMIOX"). YL-23 was either left untreated ("YL-23") or exogenously supplemented with myo-inositol ("MI") and glucuronic acid ("GlcA") (as indicated on the graph).

[0044] [Figure 26] The production of QA, QA-C3-GlcA-Gal ("QA-C3-GG") and QA-C3-GlcA-Gal-Xyl ("QA-C3-GGX"), was compared as analyzed by LC-MS. UDP-xylose synthase from Arabidopsis thaliana ("AtUXS") was overexpressed in yeast engineered to produce QA-C3-GGX under the control of the pTetOn inducible promoter. Yeast cultures were left untreated ("no inducer") or treated with various concentrations of doxycycline (as indicated on the graph).

[0045] [Figure 27]Panel A shows the comparison of UDP-xylose ("UDP-Xyl") production with UDP-fucose ("UDP-Fuc") production in various yeast strains (shown on the graph) overexpressing UDP-glucose dehydrogenase from Arabidopsis thaliana ("AtUGD"), UDP-xylose synthase from Arabidopsis thaliana ("AtUXS"), UDP-glucose-4,6-dehydratase from Saponaria vacccaria ("SvUG46DH"), UDP-4-keto-6-deoxyglucose reductase from Saponaria vacccaria ("SvNMD"), and UDP-4-keto-6-deoxyglucose reductase from Quillaja japonica in various combinations (shown on the graph). Panel B shows UDP-rhamnose synthase from Arabidopsis thaliana ("AtRHM2"), UDP-xylose synthase from Arabidopsis thaliana ("AtUXS"), UDP-glucose dehydrogenase from Arabidopsis thaliana ("AtUGD"), UDP-xylose synthase from Arabidopsis thaliana ("AtUXS"), UDP-glucose-4,6-dehydratase from Saponaria vacccaria ("SvUG46DH"), and UDP-glucose-4,6-dehydratase from Saponaria vacccaria ("SvUG46DH"). 1 shows a comparison of UDP-xylose ("UDP-Xyl"), UDP-fucose ("UDP-Fuc"), and UDP-rhamnose ("UDP-Rha") production in various yeast strains (shown on the graph) overexpressing UDP-4-keto-6-deoxyglucose reductase from S. carya ("SvNMD") and UDP-4-keto-6-deoxyglucose reductase from S. quillaja in various combinations (shown on the graph). "CP" indicates cell pellet.

[0046] [Figure 28]LC-MS extracted ion chromatograms (EICs) of the C3-glycosylated QA derivatives ("QA-C3-GlcA", "QA-C3-GlcA-Gal" and "QA-C3-GlcA-Gal-Rha") and the C28-glycosylated QA derivative ("QA-C3-GlcA-Gal-Rha-C28-Fuc") detected in yeast (YL-25) engineered to produce QA-C3-GlcA-Gal-Rha and to overexpress UDP-glucose-4,6-dehydratase from Saponaria vacccaria ("SvUG46DH"), UDP-4-keto-6-deoxyglucose reductase from Saponaria vacccaria ("SvNMD"), and UDP-fucose transferase from Quillaja japonica ("QsFucT"). Peaks corresponding to QA-C3-GlcA-Gal-Rha and QA-C3-GlcA-Gal-Rha-C28-Fuc are labeled.

[0047] [Figure 29] LC-MS extracted ion chromatograms (EICs) of C3-glycosylated QA derivatives ("QA-C3-GlcA", "QA-C3-GlcA-Gal" and "QA-C3-GlcA-Gal-Xyl") and C28-glycosylated QA derivatives ("QA-C3-GlcA-Gal-Xyl-C28-Fuc" and "QA-C3-GlcA-Xyl-Rha-C28-Fuc") detected in yeast (YL-28) engineered to produce QA-C3-GlcA-Gal-Xyl-C28-Fuc and overexpress UDP-rhamnose synthase from Arabidopsis thaliana ("AtRHM2") and UDP-rhamnose transferase from Quillaja japonica ("QsRhaT"). Peaks corresponding to QA-C3-GlcA-Gal, QA-C3-GlcA-Gal-Xyl and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha are labeled.

[0048] [Diagram 30]LC-MS extracted ion chromatograms (EICs) of C3-glycosylated QA derivatives ("QA-C3-GlcA", "QA-C3-GlcA-Gal" and "QA-C3-GlcA-Gal-Rha") and C28-glycosylated QA derivatives ("QA-C3-GlcA-Gal-Rha-C28-Fuc" and "QA-C3-GlcA-Gal-Rha-C28-Fuc") detected in yeast (YL-27) engineered to produce QA-C3-GlcA-Gal-Rha-C28-Fuc and overexpress UDP-rhamnose synthase from Arabidopsis thaliana ("AtRHM2") and UDP-rhamnose transferase from Quillaja japonica ("QsRhaT"). Peaks corresponding to QA-C3-GlcA-Gal-Rha and QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha are labeled.

[0049] [Diagram 31] The C3-glycosylated QA derivatives ("QA-C3-GlcA", "QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha") detected in yeast (YL-30) engineered to produce QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha and to overexpress UDP-xylose synthase from Arabidopsis thaliana ("AtUXS") and UDP-xylose transferase from Quillaja japonica ("QsC28XylT3") were also detected in yeast (YL-30) engineered to overexpress UDP-xylose synthase from Arabidopsis thaliana ("AtUXS") and UDP-xylose transferase from Quillaja japonica ("QsC28XylT3"). Figure 1 shows LC-MS extracted ion chromatograms (EICs) of the C28-glycosylated QA derivatives (QA-C3-GlcA-Gal, QA-C3-GlcA-Gal-Xyl, and QA-C3-GlcA-Gal-Xyl-C28-Fuc, QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha, and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl). Peaks corresponding to QA-C3-GlcA, QA-C3-GlcA-Gal, QA-C3-GlcA-Gal-Xyl, QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha, and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl are labeled.

[0050] [Diagram 32]The C3-glycosylated QA derivatives ("QA-C3-GlcA", "QA-C3-GlC-Gal-Xyl-C28-Fuc-Rha") were detected in yeast (YL-29) engineered to produce QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha and to overexpress UDP-rhamnose synthase from Arabidopsis thaliana ("AtRHM2") and UDP-rhamnose transferase from Quillaja japonica ("QsRhaT"). Figure 1 shows LC-MS extracted ion chromatograms (EICs) of QA-C3-GlcA-Gal, QA-C3-GlcA-Gal-Rha, and C28-glycosylated QA derivatives (QA-C3-GlcA-Gal-Rha-C28-Fuc, QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha, and QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl). Peaks corresponding to QA-C3-GlcA-Gal, QA-C3-GlcA-Gal-Rha, QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha, and QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl are labeled.

[0051] [Diagram 33] LC-MS extracted ion chromatograms (EICs) of C3-glycosylated QA derivatives ("QA-C3-G," "QA-C3-GG," and "QA-C3-GGX") and C28-glycosylated QA derivatives ("QA-C3-GGX-C28-FR," "QA-C3-GGX-C28-FRX," and "QA-C3-GGX-C28-FRXX") detected in yeast (YL-33) engineered to produce QA-C3-GGX-C28-FRX and overexpress UDP-xylose synthase from Arabidopsis thaliana ("AtUXS") and UDP-xylose transferase from Quillaja japonica ("QsC28XylT4"). Peaks corresponding to QA-C3-GG, QA-C3-GGX, QA-C3-GGX-C28-FR, QA-C3-GGX-C28-FRX and QA-C3-GGX-C28-FRXX are labelled.

[0052] [Diagram 34]LC-MS extracted ion chromatograms (EICs) of C3-glycosylated QA derivatives ("QA-C3-G," "QA-C3-GG," and "QA-C3-GGR") and C28-glycosylated QA derivatives ("QA-C3-GGR-C28-FR," "QA-C3-GGR-C28-FRX," and "QA-C3-GGR-C28-FRXX") detected in yeast (YL-31) engineered to produce QA-C3-GGR-C28-FRX and overexpress UDP-rhamnose synthase from Arabidopsis thaliana ("AtRHM2") and UDP-rhamnose transferase from Quillaja japonica ("QsRhaT"). Peaks corresponding to QA-C3-GG, QA-C3-GGR, QA-C3-GGR-C28-FR, QA-C3-GGR-C28-FRX and QA-C3-GGR-C28-FRXX are labelled.

[0053] [Diagram 35] LC-MS extracted ion chromatograms (EICs) of C3-glycosylated QA derivatives ("QA-C3-G," "QA-C3-GG," and "QA-C3-GGX") and C28-glycosylated QA derivatives ("QA-C3-GGX-C28-FR," "QA-C3-GGX-C28-FRX," and "QA-C3-GGX-C28-FRXA") detected in yeast (YL-34) engineered to produce QA-C3-GGX-C28-FRX and to overexpress UDP-apiose synthase from Quillaja japonica ("QsAXS") and UDP-apiose transferase from Quillaja japonica ("QsC28ApiT4"). Peaks corresponding to QA-C3-GG, QA-C3-GGX, QA-C3-GGX-C28-FR, QA-C3-GGX-C28-FRX and QA-C3-GGX-C28-FRXA are labelled.

[0054] [Diagram 36]LC-MS extracted ion chromatograms (EICs) of C3-glycosylated QA derivatives ("QA-C3-G," "QA-C3-GG," and "QA-C3-GGR") and C28-glycosylated QA derivatives ("QA-C3-GGR-C28-FR," "QA-C3-GGR-C28-FRX," and "QA-C3-GGR-C28-FRXA") detected in yeast (YL-32) engineered to produce QA-C3-GGR-C28-FRX and to overexpress UDP-apiose synthase from Quillaja japonica ("QsAXS") and UDP-apiose transferase from Quillaja japonica ("QsC28ApiT4"). Peaks corresponding to QA-C3-GG, QA-C3-GGR, QA-C3-GGR-C28-FR, QA-C3-GGR-C28-FRX and QA-C3-GGR-C28-FRXA are labeled.

[0055] [Figure 37] The figures show the results of comparing the intracellular localization of two xylose transferases derived from Quillaja japonica (QsC28XylT3-GFP and QsC28XylT4) and an apiose transferase derived from Quillaja japonica (QsC28ApiT-GFP) tagged with GFP at their C-terminus.

[0056] [Figure 38]Figure 1 shows the protein expression (measured by fluorescence intensity after flow cytometry) of various variants of QsC28XylT4 (as indicated on the graph) overexpressed in yeast engineered to produce QA-C3-GGX-C28-FRX. "QsC28XylT4-3aa", "QsC28XylT4-3aa", "QsC28XylT4-6aa", "QsC28XylT4-9aa" and "QsC28XylT4-12aa" represent variants of QsC28XylT4 with 3, 6, 9 or 12 amino acids deleted from the N-terminus, respectively. "QsC28XylT4-MBP," "QsC28XylT4-SUMO," and "QsC28XylT4-TrxA" represent variants of QsC28XylT4 with an MBP solubility tag, a SUMO solubility tag, or a TrxA solubility tag added to the N-terminus, respectively.

[0057] [Figure 39] 1 shows the results of comparing the production of QA-C3-GGX-C28-FRXX in yeast overexpressing QsC28XylT4-3aa, QsC28XylT4-6aa, QsC28XylT4-9aa, QsC28XylT4-12aa, QsC28XylT4-SUMO, QsC28XylT4-TrxA or QsC28XylT4-MBP (as indicated on the graph).

[0058] [Diagram 40] The results show a comparison of the intracellular localization of a fusion variant (QsC28XylT3-3xGGGS-QsC28XylT4), in which the two enzymes are separated by a linker (3xGGGS) and fused to the N-terminus of QsC28XylT3, and non-recombinant QsC28XylT4.

[0059] [Diagram 41]Figure 1 shows LC-MS extracted ion chromatograms (EICs) of C28-glycosylated QA derivatives ("QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha" and "QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl") detected in yeast (YL-41) engineered to produce QA-C3-GGR-C28-FRX and overexpress QsC28XylT4 (panel A) or the QsC28XylT3-3xGGGS-QsC28XylT4 fusion variant (panel B). Peaks corresponding to QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl are labeled.

[0060] [Diagram 42] Figure 1 shows the expression level of overexpressed QsC28XylT4 protein from yeast (measured by fluorescence intensity after flow cytometry analysis) at 60 hours under various cultivation conditions. Yeast cultures were grown without treatment ("control"), in the same medium supplemented with galactose or glucose ("old medium"), or in fresh medium supplemented with galactose or glucose ("fresh medium") (as indicated on the graph).

[0061] [Diagram 43] LC-MS extracted ion chromatograms (EICs) of S-2-methylbutyryl-CoA ("2MB-CoA"). The top chromatogram is from the "2M-CoA standard". The middle chromatogram is from yeast overexpressing Quillaja quinolones CoA ligase ("QsCCL") (YL-QSCCL) with 50 mg / L of 2-methylbutyrate exogenously added. The bottom chromatogram is from yeast overexpressing Aspergillus nidulans phosphopantetheinyl transferase ("AnNpgA"), Aspergillus terreus type I polyketide synthase (PKS) LovF ("AstLovF-TE") and QsCCL. Peaks corresponding to 2MB-CoA are labeled.

[0062] [Diagram 44] The acylated and / or glycosylated QA-C3-GGX-C28-FRX was produced, and detected by exogenous addition of 50 mg / L 2-methylbutyrate to yeast (YL-42) engineered to overexpress chalcone synthase-like type III polyketide synthases from Quillaja japonica ("ChsD" and "ChSE"), ketoreductases from Quillaja japonica ("KR11" and "KR23"), QsCCL, and acyltransferase from Quillaja japonica ("QsDMOT9"). LC-MS extracted ion chromatograms (EICs) of the A derivatives ("QA-C3-GGX-C28-FRX-C9", "QA-C3-GGX-C28-FRXX", "QA-C3-GGX-C28-FRXX-C9" and "QA-C3-GGX-C28-FRX" are shown. Peaks corresponding to QA-C3-GGX-C28-FRX-C9, QA-C3-GGX-C28-FRXX, QA-C3-GGX-C28-FRXX-C9, and QA-C3-GGX-C28-FRX are labeled.

[0063] [Diagram 45] FIG. 1 shows the results of comparing the production of QA-C3-GGX-C28-FRX and QA-C3-GGX-C28-FRX-C9 from YL-42 by exogenously adding increasing concentrations of 2-methylbutyric acid (as indicated on the graph).

[0064] [Diagram 46]Figure 1 shows LC-MS extracted ion chromatograms (EICs) of acylated and / or glycosylated QA derivatives ("QA-C3-GGX-C28-FRX," "QA-C3-GGX-C28-FR-C9," and "QA-C3-GGX-C28-FRXX-C18") detected by exogenous addition of 500 mg / L 2-methylbutyric acid to yeast (YL-43) engineered to produce QA-C3-GGX-C28-FRX-C9 and further overexpress an acyltransferase from Quillaja japonica ("QsDMOT4"). Peaks corresponding to QA-C3-GGX-C28-FRX, QA-C3-GGX-C28-FR-C9, and QA-C3-GGX-C28-FRXX-C18 are labeled.

[0065] [Figure 47] LC-MS extracted ion chromatograms (EICs) of acylated and / or glycosylated QA derivatives ("QA-C3-GGX-C28-FRX", "QA-C3-GGX-C28-FRX-C9", "QA-C3-GGX-C28-FRXX", "QA-C3-GGX-C28-FRXX-C9", "QA-C3-GGX-C28-FRX-C18" and "QA-C3-GGX-C28-FRXX-C18") detected by exogenous addition of 500 mg / L 2-methylbutyric acid to yeast (YL-44) engineered to produce QA-C3-GGX-C28-FRXX-C9 and further overexpress an acyltransferase from Quillaja japonica ("QsDMOT4"). Peaks corresponding to QA-C3-GGX-C28-FRX, QA-C3-GGX-C28-FRX-C9, QA-C3-GGX-C28-FRXX-C9, and QA-C3-GGX-C28-FRXX-C18 are labeled.

[0066] [Figure 48]Panel A shows the comparison of UDP-arabinopyranose ("UDP-Arap") and UDP-arabinofuranose ("UDP-Araf") production in various yeast strains overexpressing arabinokinase from Arabidopsis thaliana ("AtAraK"), arabinokinase from pathogenic Leptospira interrogans (Lei) ("LeiAraK"), UDP-sugar pyrophosphorylase from Arabidopsis thaliana ("AtUSP"), UDP-sugar pyrophosphorylase from pathogenic Leptospira ("LeiUSP"), arabinose transporter from Penicillium rubens Wisconsin ("PrAraT"), and UDP-arabinose mutase ("AtUAM1") in various combinations (as indicated on the graph). Panel B shows the comparison of UDP-arabinopyranose ("UDP-Arap") and UDP-xylose ("UDP-Xyl") production in various yeast strains overexpressing UDP-arabinose mutase from Arabidopsis thaliana ("AtUAM1"), UDP-arabinose mutase from H. vulgare ("HvUAM"), two UDP-xylose epimerases UXE from Arabidopsis thaliana ("AtUXE" or "AtUXE2"), and UDP-glucose 4-epimerase from Arabidopsis thaliana ("AtUGE3") in various combinations (as indicated on the graph). "CP" indicates cell pellet.

[0067] [Figure 49]Acylation and / or glycosylation detected by exogenous addition of 500 mg / L 2-methylbutyrate to yeast (YL-45) engineered to produce QA-C3-GGX-C28-FRXX-C18 and further overexpress UDP-xylose epimerase from Arabidopsis thaliana ("AtUXE") and UDP-arabinose mutase from Arabidopsis thaliana ("AtUAM1"). Figure 1 shows LC-MS extracted ion chromatograms (EICs) of the purified QA derivatives ("QA-C3-GGX-C28-FRX," "QA-C3-GGX-C28-FRXX-C9," "QA-C3-GGX-C28-FRX-C18," "QA-C3-GGX-C28-FRX-C18-Araf," and "QS-21-Xyl," corresponding to QA-C3-GGX-C28-FRX-C18-Araf). Peaks corresponding to QA-C3-GGX-C28-FRX, QA-C3-GGX-C28-FRX-C9, QA-C3-GGX-C28-FRXX-C9, and QA-C3-GGX-C28-FRXX-C18 are labeled.

[0068] [Figure 50] The LC-MS extracted ion chromatogram (EIC) of "QS-21-Xyl" (corresponding to QA-C3-GGX-C28-FRXX-C18-Araf) is shown. When compared with a QS-21 standard (a QS-21 fraction purified from Quillaja bark), it was observed that the two peaks matched (inset).

[0069] [Figure 51]Acylated and / or glycosylated QA-C3-GGX-C28-FRXA-C18 was produced and detected by exogenous addition of 500 mg / L 2-methylbutyrate to yeast (YL-46) engineered to overexpress UDP-xylose epimerase from Arabidopsis thaliana ("AtUXE"), UDP-arabinose mutase from Arabidopsis thaliana ("AtUAM1"), and arabinofuranose transferase from Quillaja japonica ("QsArafT"). LC-MS extracted ion chromatograms (EICs) of the A derivatives ("QA-C3-GGX-C28-FRX", "QA-C3-GGX-C28-FRXA", "QA-C3-GGX-C28-FRX-C9", "QA-C3-GGX-C28-FRXX-C9", "QA-C3-GGX-C28-FRX-C18", "QA-C3-GGX-C28-FRXX-C18-Araf", and "QS-21-Api" corresponding to QA-C3-GGX-C28-FRX-C18-Araf) are shown. Peaks corresponding to QA-C3-GGX-C28-FRX, QA-C3-GGX-C28-FRX-C9, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18-Araf, and QS-21-Api are labeled.

[0070] [Figure 52] LC-MS extracted ion chromatograms (EICs) of acylated and / or glycosylated QA derivatives ("QA-C3-GGX-FRX," "QA-C3-GGX-C28-FRX-C9," "QA-C3-GGX-C28-FRX-C18," and "QA-C3-GGX-C28-FRX-C18-Xyl") detected by exogenous addition of 500 mg / L 2-methylbutyric acid to yeast (YL-47) engineered to produce QA-C3-GGX-C28-FRX-C18 and further overexpress arabinofuranose transferase from Quillaja japonica ("QsArafT"). Peaks corresponding to QA-C3-GGX-C28-FRX-C9, QA-C3-GGX-C28-FRX-C18, and QA-C3-GGX-C28-FRX-C18-Xyl are labeled.

[0071] [Diagram 53] Figure 1 shows LC-MS extracted ion chromatograms (EICs) of acylated glycosylated QA derivatives ("QA-C3-GGX-FRX-C9," "QA-C3-GGX-C28-FRX-C18," and "QA-C3-GGX-C28-FRX-C18-Xyl") detected by exogenous addition of 500 mg / L 2-methylbutyric acid to yeast (YL-48) engineered to produce QA-C3-GGX-C28-FRX-C18 and additionally overexpress arabinofuranose transferase from Quillaja japonica ("QsArafT2"). Peaks corresponding to QA-C3-GGX-FRX-C9 and QA-C3-GGX-C28-FRX-C18 are labeled.

[0072] [Figure 54] LC-MS extracted ion chromatograms (EICs) of acylated glycosylated QA derivatives ("QA-C3-GGX-FRX-C9", "QA-C3-GGX-C28-FRX-C18", "QA-C3-GGX-C28-FRX-C18-Araf" and "QA-C3-GGX-C28-FRXX-C18-Araf") detected by exogenous addition of 500 mg / L 2-methylbutyric acid to yeast (YL-49) engineered to produce QA-C3-GGX-C28-FRXX-C18 and further overexpress arabinofuranose transferase from Quillaja japonica ("QsArafT2"). Peaks corresponding to QA-C3-GGX-FRX-C9, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18-Araf, and QA-C3-GGX-C28-FRXX-C18-Araf are labeled.

[0073] [Figure 55]LC-MS extracted ion chromatograms (EICs) of acylated glycosylated QA derivatives ("QS-21-Xyl", "QA-C3-GGX-FRX-C9", "QA-C3-GGX-C28-FRXX-C9", "QA-C3-GGX-C28-FRX-C18" and "QA-C3-GGX-C28-FRX-C18-Araf") detected without exogenous addition of 2-methylbutyrate in yeast (YL-50) engineered to produce QA-C3-GGX-C28-FRXX-C18-Araf and further overexpress phosphopantetheinyl transferase from Aspergillus nidulans ("AnNpgA") and type I polyketide synthase (PKS) LovF from Aspergillus terreus ("AstLovF-TE"). Peaks corresponding to QS-21-Xyl, QA-C3-GGX-FRX-C9, QA-C3-GGX-C28-FRX-C18, and QA-C3-GGX-C28-FRX-C18-Araf are labeled.

[0074] [Figure 56] The acylated glycosylated QA derivative (QA-C3-GGX-C28-FRXX-C18-Araf) was produced and detected without exogenous addition of 2-methylbutyrate in yeast (YL-51) engineered to overexpress phosphopantetheinyl transferase from Aspergillus nidulans ("AnNpgA") and the type I polyketide synthase (PKS) LovF ("AstLovF-TE"). Figure 1 shows LC-MS extracted ion chromatograms (EICs) of "QS-21-Api," "QA-C3-GGX-FRXA," "QA-C3-GGX-C28-FRX-C9," "QA-C3-GGX-C28-FRXA-C9," and "QA-C3-GGX-C28-FRX-C18" corresponding to QS-21-Api, QA-C3-GGX-C28-FRX-C, and QA-C3-GGX-C28-FRX-C18. Peaks corresponding to QS-21-Api, QA-C3-GGX-C28-FRX-C, and QA-C3-GGX-C28-FRX-C18 are labeled.

[0075] [Figure 57]LC-MS extracted ion chromatograms (EICs) of “QA-C3-GlcA-C28-Fuc” detected in N. benthamiana plants transiently co-expressing various combinations (as shown) of UDP-glucuronosyltransferase from Saponaria vacccaria (“SvCslG”), UDP-glucose-4,6-dehydratase from Saponaria vacccaria (“SvUG46DH”), UDP-4-keto-6-deoxyglucose reductase from Saponaria vacccaria (“SvNMD”), fucose transferase from Quillaja japonica (“QsFucT”), fucose transferase from Saponaria vacccaria (“SvFucT”), and “GFP” (used as a negative control) were loaded with QA (commercially available). The peak corresponding to QA-C3-GlcA-C28-Fuc is labelled.

[0076] [Figure 58] LC-MS extracted ion chromatogram (EIC) of "QA-C3-GlcA-Gal-Xyl" detected in N. benthamiana plants transiently co-expressing Saponaria vacccaria UDP-glucuronosyltransferase ("SvCslG"), Saponaria vacccaria galactosetransferase ("SvGalT"), and Saponaria vacccaria xylosetransferase ("SvC3XylT") when loaded with QA (commercially available). The peak corresponding to QA-C3-GlcA-Gal-Xyl is labeled. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The inventors have succeeded in reconstructing for the first time a metabolic pathway capable of biosynthesizing QS-21 from the monosaccharide galactose in Saccharomyces cerevisiae by using more than 30 heterologous proteins from various plants and microorganisms belonging to six distinct protein classes: terpene synthases, cytochrome P450 monooxygenases (i.e., "CYP oxidases"), nucleotide sugar synthases, sugar transferases, acyltransferases, and polyketide synthases (PKSs).

[0078] The triterpene core of QS-21, quilacic acid (QA), is derived from the simple triterpene β-amyrin, which is synthesized by cyclization of the versatile linear precursor 2,3-oxidosqualene (OS) (along the mevalonate pathway naturally occurring in yeast (Wong et al., 2018)) by oxidosqualene cyclase (OSC), also known as β-amyrin synthase (BAS) (Figure 2B). The C28, C16, and C23 positions of this scaffold β-amyrin are sequentially oxidized by three CYP oxidases to form a carboxylic acid, an alcohol, and an aldehyde, respectively, to generate quilacic acid (QA) (Figure 2B).

[0079] Next, UDP-glucuronic acid ("UDP-GlcA"), UDP-galactose ("UDP-Gal"), and UDP-xylose ("UDP-Xyl") or UDP-rhamnose ("UDP-Rha") are incorporated at the C3 position of QA by the corresponding glycosyltransferases to generate C3-glycosylated QA derivatives (see FIG. 2C). In the present specification, such C3-glycosylated QA derivatives are respectively represented as follows: QA-C3-GlcA or QA-C3-G QA-C3-GlcA-Gal or QA-C3-GG QA-C3-GlcA-Gal-Rha or QA-C3-GGR QA-C3-GlcA-Gal-Xyl or QA-C3-GGX Table 1 shows these chemical formulas.

[0080] Next, UDP-fucose ("UDP-Fuc"), UDP-Rha, UDP-Xyl, and a second UDP-Xyl or UDP-Api are incorporated at the C28 position of QA by the corresponding glycosyltransferases to generate C28-glycosylated QA derivatives (see FIG. 2D). In the present specification, such C28-glycosylated QA derivatives are respectively represented as follows: QA-C3-GlcA-Gal-Xyl-C28-Fuc or QA-C3-GGX-C28-F QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha or QA-C3-GGX-C28-FR ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl or QA-C3-GGX-C28-FRX ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GGX-C28-FRXX ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api or QA-C3-GGX-C28-FRXA QA-C3-GlcA-Gal-Rha-C28-Fuc or QA-C3-GGR-C28-F ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GGR-C28-FR ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GGR-C28-FRX ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GGR-C28-FRXX ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GGR-C28-FRXA These chemical formulas are shown in Table 1.

[0081] Biosynthesis of the 18-carbon pseudodimeric acyl chain is accomplished by condensing malonyl-CoA (naturally occurring in yeast) with S-2-methylbutyryl-CoA ("2MB-CoA") to generate C9-CoA using a type I polyketide synthase ("PKS"), a carboxyl-coenzyme A ligase ("CCL"), a type III PKS, and a ketoreductase (KR) (see Figure 2A).

[0082] Two repeating C9-CoA acyl units are then sequentially transferred by two acyltransferases to add an 18-carbon pseudodimeric acyl chain to the fucose residue at C28 of the linear tetrasaccharide to generate acylated glycosylated QA derivatives, which are referred to herein as follows: ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-C9 or QA-C3-GGX-C28-FRX-C9 ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl-C9 or QA-C3-GGX-C28-FRXX-C9 ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api-C9 or QA-C3-GGX-C28-FRXA-C9 ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-C9 or QA-C3-GGR-C28-FRX-C9 ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl-C9 or QA-C3-GGR-C28-FRXX-C9 ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api-C9 or QA-C3-GGR-C28-FRXA-C9 ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-C18 or QA-C3-GGX-C28-FRX-C18 ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl-C18 or QA-C3-GGX-C28-FRXX-C18 ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api-C18 or QA-C3-GGX-C28-FRXA-C18 ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-C18 or QA-C3-GGR-C28-FRX-C18 ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl-C18 or QA-C3-GGR-C28-FRXX-C18 ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api-C18 or QA-C3-GGR-C28-FRXA-C18 These chemical formulas are shown in Table 1.

[0083] UDP-arabinofuranose ("UDP-Araf") or UDP-Xyl are then incorporated at the terminus of the 18-carbon pseudodimeric acyl chain (i.e., at the 5-hydroxy functionality of the second C9-CoA acyl unit) to generate further acylated and glycosylated QA derivatives (see Figure 2E). The further acylated and glycosylated QA derivatives include the two major QS-21 isomers present in the conventionally purified QS-21 fraction from Quillaja bark and their rhamnose-derived species (see Figure 1). These acylated and glycosylated QA derivatives are referred to herein as follows: ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-C18-Araf or QA-C3-GGX-C28-FRX-C18-Araf QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl-C18-Araf or QA-C3-GGX-C28-FRXX-C18-Araf or QS-21-Xyl ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf or QS-21-Api ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-C18-Araf or QA-C3-GGR-C28-FRX-C18-Araf ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl-C18-Araf or QA-C3-GGR-C28-FRXX-C18-Araf ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api-C18-Araf or QA-C3-GGR-C28-FRXA-C18-Araf ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-C18-Xyl or QA-C3-GGX-C28-FRX-C18-Xyl ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl-C18-Xyl or QA-C3-GGX-C28-FRXX-C18-Xyl ·QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api-C18-Xyl or QA-C3-GGX-C28-FRXA-C18-Xyl ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl-C18-Xyl or QA-C3-GGR-C28-FRXX-C18-Araf ·QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api-C18-Xyl or QA-C3-GGR-C28-FRXA-C18-Xyl These chemical formulas are shown in Table 1.

[0084] In the context of the present invention, a "C3-glycosylated QA derivative" is a QA derivative that contains at least a glucuronic acid residue at position C3 (see list above). In the context of the present invention, a "C28-glycosylated QA derivative" is a QA derivative that contains all sugars at position C3 of the branched trisaccharide and at least a fucose residue at position C28 of the linear tetrasaccharide (see list above). In the context of the present invention, an "acylated glycosylated QA derivative" is a QA derivative that contains all sugars at position C3 of the branched trisaccharide, at least the first three sugars at position C28 of the linear tetrasaccharide, at least one C9-CoA acyl unit ("C9") bound to a fucose residue, and may contain an arabinofuranose residue if two C9-CoA acyl units (i.e. "C18") are attached (see list above).

[0085] In the context of the present invention, a "heterologous gene" is a gene that is not naturally expressed in yeast.

[0086] In the context of the present invention, for example, a "yeast recombinantly engineered to produce a sugar or QA derivative" is a yeast which overexpresses a heterologous gene encoding an enzyme or protein required for the biosynthesis or production of the respective QA derivative, as described, for example, in each of the methods according to the first to tenth aspects of the present invention.

[0087] Preparation of Quillaric Acid (QA) and Its Optimization WO19 / 122259 reports the identification of enzymes involved in the biosynthesis of QA from the Quillaja genome and the production of QA in Nicotiana benthamiana recombinantly modified with such enzymes. WO20 / 263524 also reports the tracing of QA production in yeast recombinantly modified with enzymes from various plants. The contents of WO19 / 122259 and WO20 / 263524 are both incorporated herein by reference.

[0088] β-amyrin The first step of the method according to the first aspect of the present invention is the formation of β-amyrin by cyclization of 2,3-oxidosqualene. This step is carried out by oxidosqualene cyclase or β-amyrin synthase (BAS). Any heterologous plant-derived β-amyrin synthase capable of producing β-amyrin can be suitably used in the method of the present invention. For example, a β-amyrin synthase (BAS) from Artemisia annua (A. annua or "Aa"), Arabidopsis thaliana (A. thaliana or "At"), Glycyrrhiza glabra (G. glabra or "gG"), Gypsophila vaccaria (G. vaccaria or "Gv"), Medicago truncatula (M. truncatula or "Mt"), Quillaja saponaria (Q. saponaria or "Q"), or Saponaria vaccaria (S. vaccaria or "Sv") may be used. In some embodiments, the method according to the first aspect of the invention uses a β-amyrin synthase from a plant selected from the above plants. Specifically, the β-amyrin synthesis enzyme may be selected from AaBAS as set forth in SEQ ID NO: 1, AtBAS as set forth in SEQ ID NO: 4, GgBAS as set forth in SEQ ID NO: 7, GvBAS as set forth in SEQ ID NO: 10, QsBAS as set forth in SEQ ID NO: 15 and SvBAS as set forth in SEQ ID NO: 13. Advantageously, the β-amyrin synthesis enzyme is GvBAS as set forth in SEQ ID NO: 10.

[0089] Alternatively, AaBAS, AtBAS, GgBAS, GvBAS, GvBAS, QsBAS or SvBAS may be a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:15 or SEQ ID NO:13.

[0090] Kira Acid (QA) As previously described, QA is generated by sequential oxidation of the C28, C16, and C23 positions of β-amyrin by the corresponding cytochrome P450 (CYP) oxidases to form carboxylic acid, hydroxyl, and aldehyde groups, respectively.

[0091] Any heterologous plant-derived CYP oxidase that has been previously identified and reported to be capable of efficiently functionalizing the C28, C16, and C23 positions of β-amyrin may be used in the methods and recombinant yeast of the present invention (e.g., those described and reported in WO19 / 122259 or WO2020 / 263524, or in the review by Gosh (2017), the contents of which are incorporated herein by reference). In some embodiments, the method according to the first aspect of the invention uses a CYP C16 oxidase, a CYP C23 oxidase, and a CYP C28 oxidase independently selected from those derived from A. annua, A. thaliana, G. glabra, M. truncatula, Q. saponaria, S. vaccaria, Centella asiatica, Bupleurum falcatum, Maesa lanceolata, Q. saponaria, and S. vaccaria.

[0092] In further embodiments, the CYP C16 oxidase is selected from CYP87D16 and CYP716Y1; the CYP C23 oxidase is selected from CYP72A68 and CYP714E19; and The C28 oxidase is selected from CYP716A1, CYP716A12, CYP716A15, CYP716A17, CYP716A44, CYP716A46, CYP716A52v2, CYP716A75, CYP716A78, CYP716A79, CYP716A80, CYP716A81, CYP716A83, CYP716A86, CYP716A110, CYP716A140, CYP716A179, CYP716A252, CYP16A253, and CYP716AL1.

[0093] In further embodiments, the CYP C16 oxidase is selected from BfC16 as set forth in SEQ ID NO:17, QsC16 oxidase as set forth in SEQ ID NO:20, QsC28C16 as set forth in SEQ ID NO:23, and SvC16 as set forth in SEQ ID NO:26.

[0094] In a further embodiment, the CYP C16 oxidase is selected from BfC16 as set forth in SEQ ID NO: 17, SvC16 as set forth in SEQ ID NO: 26, QsC16 as set forth in SEQ ID NO: 20, and QsC28C16 as set forth in SEQ ID NO: 23. Alternatively, BfC16, SvC16, QsC16 and QsC28C16 may be set forth in sequences having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 20 and SEQ ID NO: 23, respectively.

[0095] In a further embodiment, the CYP C23 oxidase is selected from MtC23 oxidase as set forth in SEQ ID NO: 38, QsC23 as set forth in SEQ ID NO: 29, SvC23-1 as set forth in SEQ ID NO: 32, and SvC23-2 as set forth in SEQ ID NO: 35. Alternatively, MtC23, QsC23, SvC23-1 and SvC23-2 may be set forth in sequences having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 38, SEQ ID NO: 29, SEQ ID NO: 32 and SEQ ID NO: 35, respectively.

[0096] In a further embodiment, the CYP C28 oxidase is selected from MtC28 as set forth in SEQ ID NO: 46, QsC28 as set forth in SEQ ID NO: 41, and SvC28 as set forth in SEQ ID NO: 44. Alternatively, MtC28, QsC28 and SvC28 may be set forth in sequences having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 46, SEQ ID NO: 41 and SEQ ID NO: 44, respectively.

[0097] In the method according to the first aspect of the invention, a heterologous redox partner, such as a cytochrome P450 reductase (CPR) and / or a cytochrome b5, may be further co-expressed. For example, the CPR may be selected from those derived from Arabidopsis thaliana and Lotus japonicus. In some embodiments, the CPR is selected from AtATR1 as set forth in SEQ ID NO: 49 and Lj CPR as set forth in SEQ ID NO: 52.

[0098] The heterologous cytochrome b5 may be selected from those derived from Arabidopsis thaliana, Quillaja japonica, and Saponaria vaccaria. In some embodiments, the cytochrome b5 is selected from Atb5 as set forth in SEQ ID NO:58, Qsb5 as set forth in SEQ ID NO:55, and Svb5 as set forth in SEQ ID NO:61. Alternatively, Atb5, Qsb5, and Svb5 may be those set forth in sequences having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:58, SEQ ID NO:55, and SEQ ID NO:61, respectively.

[0099] In the method according to the first aspect of the invention, a heterologous scaffold protein (allowing the P450 enzymes to physically assemble) may further be co-expressed. The scaffold protein may be a membrane steroid binding protein (MSBP). For example, the MSBP may be selected from those derived from Arabidopsis thaliana, Quillaja japonica and Saponaria vaccaria. In some embodiments, the MSBP is selected from AtMSBP1 as set forth in SEQ ID NO:63, AtMSBP2 as set forth in SEQ ID NO:65, QsMSBP1 as set forth in SEQ ID NO:73, SvMSBP1 as set forth in SEQ ID NO:67, and SvMSBP2 as set forth in SEQ ID NO:70. Alternatively, AtMSBP2, QsMSBP1, SvMSBP1 and SvMSBP2 may be those set forth in sequences having at least 70%, 80%, 90%, 95%, 98% or 99% identity with SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:67 and SEQ ID NO:70, respectively.

[0100] The first aspect of the invention further provides a yeast engineered to produce QA.

[0101] Generation of C3-glycosylated QA derivatives As mentioned above, a branched trisaccharide consisting of GlcA, Gal and Xyl (or Rha) is attached to the C3 position of QA.

[0102] Production of unnatural sugars The method according to the second aspect of the invention comprises overexpressing a heterologous gene encoding a UDP-glucose dehydrogenase (UGD) that converts UDP-glucose (UDP-Glc) to UDP-GlcA. UGDs from various plants may be used. In some embodiments, the UGD is selected from those derived from Arabidopsis thaliana, Synechococcus (Syn), Homo sapiens (Hs), Paramoeba atlantica (Patl), Bacillus cytotoxicus (Bcyt), Corallococcus macrosporus (Myxfulv), and Pyrococcus furiosus (Pfu). In further embodiments, the UGD is selected from AtUGD as set forth in SEQ ID NO: 84, AtUGD as set forth in SEQ ID NO: 108, AtUGD as set forth in SEQ ID NO: 109, AtUGD as set forth in SEQ ID NO: 110, AtUGD as set forth in SEQ ID NO: 111, AtUGD as set forth in SEQ ID NO: 112, AtUGD as set forth in SEQ ID NO: 113, AtUGD as set forth in SEQ ID NO: 114, AtUGD as set forth in SEQ ID NO: 115, AtUGD as set forth in SEQ ID NO: 116, AtUGD as set forth in SEQ ID NO: 117, AtUGD as set forth in SEQ ID NO: 118, AtUGD as set forth in SEQ ID NO: 119, AtUGD as set forth in SEQ ID NO: 120, AtUGD as set forth in SEQ ID NO: 121, AtUGD as set forth in SEQ ID NO: 122, AtUGD as set forth in SEQ ID NO: 123, AtUGD as set forth in SEQ ID NO: 124, AtUGD as set forth in SEQ ID NO: 125, AtUGD as set forth in SEQ ID NO: A101L , SynUGD as shown in SEQ ID NO: 154, HsUGD as shown in SEQ ID NO: 157 A104L , PatlUGD as set forth in SEQ ID NO: 110, BcytUGD as set forth in SEQ ID NO: 160, MyxfulvUGD as set forth in SEQ ID NO: 163, and PfuUGD as set forth in SEQ ID NO: 166. A101L , SynUGD, HsUGD A104L , PatlUGD, BcytUGD, MyxfulvUGD, and PfuUGD may be sequences having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:84, SEQ ID NO:108, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:110, SEQ ID NO:160, SEQ ID NO:163 and SEQ ID NO:166, respectively.

[0103] The second aspect of the invention further provides a yeast engineered to produce UDP-GlcA.

[0104] The first step of the method according to the third aspect of the invention is the overexpression of a heterologous gene encoding a UDP-rhamnose synthetase. UDP-rhamnose synthetases from various plants may be used. In some embodiments, the UDP-rhamnose synthetase is AtRHM2 from Arabidopsis thaliana as shown in SEQ ID NO: 102 or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 102.

[0105] The third aspect of the invention further provides a yeast engineered to produce UDP-Rha.

[0106] The first step of the method according to the fourth aspect of the invention is the overexpression of a heterologous gene encoding a UDP-glucose dehydrogenase (UGD) that converts UDP-glucose (UDP-Glc) to UDP-GlcA. The UGD may be any of the UGDs described above in the method according to the second aspect of the invention. The second step of the method according to the fourth aspect of the invention is the overexpression of a heterologous gene encoding a UDP-xylose synthase (UXS). UDP-Xyl may be produced by decarboxylation of UDP-GlcA by UDP-Xyl synthase (UXS) and / or UDP-Api / Xyl dual synthase (AXS). UDP-xylose synthase and UDP-Api / Xyl dual synthase may be derived from various plants, for example, from Arabidopsis thaliana or from Quillaja japonica. In some embodiments, the UXS is selected from AtUXS encoded by SEQ ID NO:105, QsAXS encoded by SEQ ID NO:113, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:105, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:113.

[0107] The fourth aspect of the invention further provides a yeast engineered to produce UDP-Xyl.

[0108] Generation of QA-C3-GlcA As shown in FIG. 2C, UDP-GlcA is transferred by glucuronosyltransferase (GlcAT) to attach a GlcA residue to the C3 position of QA. The first step of the method according to the fifth aspect of the invention is the overexpression of a heterologous gene encoding glucuronosyltransferase (GlcAT) in a yeast engineered to produce QA and UDP-GlcA. The yeast engineered to produce QA may be the yeast of the first aspect of the invention. The yeast engineered to produce UDP-GlcA may be the yeast of the second aspect of the invention. The GlcAT may be from any plant, for example selected from Quillaja and Saponaria vaccaria. In some embodiments, the GlcAT is selected from QsCslG1 set forth in SEQ ID NO: 78, QsCslG2 set forth in SEQ ID NO: 81, SvCslG set forth in SEQ ID NO: 76, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 78, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 81, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 76.

[0109] The fifth aspect of the invention further provides a yeast engineered to produce QA-C3-GlcA (aspect 5a).

[0110] Generation of QA-C3-GlcA-Gal As shown in FIG. 2C, UDP-Gal is transferred by galactose transferase (GalT) to attach a Gal residue to the C3 position of QA. The second step of the method according to the fifth aspect of the invention is the overexpression of a heterologous gene encoding galactose transferase (GalT) in a yeast engineered to produce QA-C3-GlcA. The yeast engineered to produce QA-C3-GlcA may be a yeast according to the fifth aspect of the invention. GalT may be from any plant, for example selected from Quillaja and Saponaria vaccaria. In some embodiments, the GalT is selected from QsGalT as set forth in SEQ ID NO:116, SvGalT as set forth in SEQ ID NO:98, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:116, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:98.

[0111] The fifth aspect of the invention further provides a yeast engineered to produce QA-C3-GlcA-Gal (aspect 5b).

[0112] Generation of QA-C3-GlcA-Gal-Rha As shown in Figure 2C, UDP-Rha is transferred by rhamnose transferase (RhaT) to attach the Rha residue to the C3 position of QA. The third step of the method according to the fifth aspect of the invention is the overexpression of a heterologous gene encoding rhamnose transferase (RhaT) in a yeast engineered to produce QA-C3-GlcA-Gal and UDP-Rha. The yeast engineered to produce QA-C3-GlcA-Gal may be a yeast according to the fifth aspect of the invention. The yeast engineered to produce UDP-Rha may be a yeast according to the third aspect of the invention.

[0113] The RhaT may be from any plant, for example from Quillaja quinata, hi some embodiments, the RhaT is QsRhaT as set forth in SEQ ID NO: 119, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 119.

[0114] The fifth aspect of the invention further provides a yeast engineered to produce QA-C3-GlcA-Gal-Rha (aspect 5c).

[0115] Generation of QA-C3-GlcA-Gal-Xyl As shown in FIG. 2C, UDP-Xyl is transferred by xylosyltransferase (XylT) to attach a Xyl residue to the C3 position of QA. Another embodiment of the third step of the method according to the fifth aspect of the invention is the overexpression of a heterologous gene encoding xylosyltransferase (XylT) in a yeast engineered to produce QA-C3-GlcA-Gal and UDP-Xyl. The yeast engineered to produce QA-C3-GlcA-Gal may be a yeast according to the fifth aspect of the invention. The yeast engineered to produce UDP-Xyl may be a yeast according to the fourth aspect of the invention. XylT may be from any plant, for example from Quillaja or Saponaria vaccaria. In some embodiments, XylT is selected from QsC3XylT as set forth in SEQ ID NO: 122, SvC3XylT as set forth in SEQ ID NO: 100, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 122, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 100.

[0116] The fifth aspect of the invention further provides a yeast engineered to produce QA-C3-GlcA-Gal-Xyl (aspect 5d).

[0117] Generation of C28-glycosylated QA derivatives As described above, a linear trisaccharide consisting of FRXX / A is linked to the C28 position of QA.

[0118] Generation of UDP-Fuc The first step of the method according to the sixth aspect of the invention is the overexpression of a heterologous gene encoding a UDP-glucose-4,6-dehydratase (UG46DH) that converts UDP-Glc to UDP-4-keto-6-deoxyglucose and / or a 4-ketoreductase that converts UDP-4-keto-6-deoxyglucose to UDP-D-Fuc. The UG46DH and the 4-ketoreductase may be from any plant, for example, independently selected from those from Quillaja japonica and Saponaria vaccaria. In some embodiments, the UG46DH is SvUG46DH as set forth in SEQ ID NO:87 or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:87. In some embodiments, the 4-keto reductase is selected from svNMD set forth in SEQ ID NO:90, QsFucSyn set forth in SEQ ID NO:175, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:90, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:175.

[0119] The sixth aspect of the invention further provides a yeast engineered to produce UDP-Fuc.

[0120] Generation of QA-C3-GGX-C28-F and QA-C3-GGR-C28-F As shown in FIG. 2D, UDP-Fuc is transferred by fucose transferase (FucT) to attach a Fuc residue to the C28 position of QA. The first step of the method according to the seventh aspect of the invention is the overexpression of a heterologous gene encoding fucose transferase (FucT) in yeast engineered to produce QA-C3-GlcA-Gal-Rha or in yeast engineered to produce QA-C3-GlcA-Gal-Xyl and UDP-fucose. The yeast engineered to produce QA-C3-GlcA-Gal-Rha may be a yeast according to the 5c aspect of the invention. The yeast engineered to produce QA-C3-GlcA-Gal-Xyl may be a yeast according to the fifth aspect of the invention. The yeast engineered to produce UDP-Fuc may be a yeast according to the fifth aspect of the invention. The FucT may be selected from those derived from Quillaja and Saponaria vaccaria. In some embodiments, the FucT is selected from QsFucT as set forth in SEQ ID NO:93, SvFucT as set forth in SEQ ID NO:96, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:93, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:96.

[0121] The seventh aspect of the invention further provides a yeast engineered to produce QA-C3-GGR-C28-F or QA-C3-GGX-C28-F (aspect 7a).

[0122] Generation of QA-C3-GGX-C28-FR and QA-C3-GGR-C28-FR As shown in Figure 2D, UDP-Rha is transferred by Rha transferase (RhaT) to attach the Rha residue to the C28 position of QA. The second step of the method according to the seventh aspect of the invention is the overexpression of a heterologous gene encoding rhamnose transferase (RhaT) in the yeast engineered to produce QA-C3-GGR-F or QA-GGX-F. The yeast engineered to produce QA-C3-GGR-F or QA-C3-GGX-F may be the yeast of aspect 7a of the invention. RhaT may be the same as that described in the method according to the fifth aspect of the invention.

[0123] The seventh aspect of the invention further provides a yeast engineered to produce QA-C3-GGR-C28-FR or QA-C3-GGX-C28-FR (aspect 7b).

[0124] Generation of QA-C3-GGX-C28-FRX and QA-C3-GGR-C28-FRX As shown in FIG. 2D, UDP-Xyl is transferred by xylosyltransferase (XylT) to attach a Xyl residue to the C28 position of QA. The third step of the method according to the seventh aspect of the invention is overexpression of a heterologous gene encoding xylosyltransferase (XylT) in yeast engineered to produce QA-C3-GGR-FR or QA-GGX-FR. The yeast engineered to produce QA-C3-GGR-FR or QA-C3-GGX-FR may be a yeast according to aspect 7b of the invention. XylT may be selected from those derived from Quillaja and Saponaria vaccaria. In some embodiments, XylT is QsC28XylT3 as set forth in SEQ ID NO: 125, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 125.

[0125] The seventh aspect of the invention further provides a yeast engineered to produce QA-C3-GGR-C28-FRX or QA-C3-GGX-C28-FRX (aspect 7c).

[0126] Generation of QA-C3-GGX-C28-FRXX and QA-C3-GGR-C28-FRXX As shown in FIG. 2D, UDP-Xyl is transferred by xylosyltransferase (XylT) to attach a Xyl residue to the C28 position of QA. The fourth step of the method according to the seventh aspect of the invention is the overexpression of a heterologous gene encoding xylosyltransferase (XylT) in the yeast engineered to produce QA-C3-GGR-FRX or QA-GGX-FRX. The yeast engineered to produce QA-C3-GGR-FRX or QA-C3-GGX-FRX may be a yeast according to aspect 7c of the invention. XylT may be from Quillaja. In some embodiments, XylT is QsC28XylT4 as shown in SEQ ID NO: 128, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 128. In further embodiments, XylT is selected from QsC28XylT4-3aa as set forth in SEQ ID NO: 131, QsC28XylT4-6aa as set forth in SEQ ID NO: 134, QsC28XylT4-9aa as set forth in SEQ ID NO: 137, QsC28XylT4-12aa as set forth in SEQ ID NO: 140, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 131, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 134, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 137, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 140. In a further embodiment, the XylT is selected from SUMO-QsC28XylT4 as set forth in SEQ ID NO: 143, TrxA-QsC28-XylT4 as set forth in SEQ ID NO: 145, and MBP-QsC28XylT4 as set forth in SEQ ID NO: 147. In a further embodiment, the XylT is QsC28XylT3-3xGGGS-QsC28XylT4 as set forth in SEQ ID NO: 149.

[0127] The seventh aspect of the invention further provides a yeast engineered to produce QA-C3-GGR-C28-FRXX or QA-C3-GGX-C28-FRXX (aspect 7d).

[0128] Generation of QA-C3-GGX-C28-FRXA and QA-C3-GGR-C28-FRXA As shown in FIG. 2D, UDP-Api is transferred by apiose transferase (XylT) to attach an Api residue to C28 of QA. Another embodiment of the fourth step of the method according to the seventh aspect of the invention is the overexpression of heterologous genes encoding UDP-apiose synthase (AXS) and / or apiose transferase (ApiT) that convert UDP-GlcA to UDP-Api in yeast engineered to produce QA-C3-GGR-FRX or QA-C3-GGX-FRX. The yeast engineered to produce QA-C3-GGR-FRX or QA-C3-GGX-FRX may be a yeast according to aspect 7c of the invention. ApiT and AXS may be independently selected from those derived from Quillaja and Saponaria vaccaria. In some embodiments, AXS is QsAXS as set forth in SEQ ID NO: 113, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 113. In some embodiments, ApiT is QsC28ApiT4 as set forth in SEQ ID NO: 151, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 151.

[0129] The seventh aspect of the invention further provides a yeast engineered to produce QA-C3-GGR-C28-FRXA or QA-C3-GGX-C28-FRXA (aspect 7e).

[0130] Formation and binding of 18-carbon pseudodimeric acyl chains (C18-Araf) terminally modified with arabinofuranose As shown in Figure 2A, biosynthesis of the 18-carbon pseudodimeric acyl chain is accomplished by condensing malonyl-CoA with 2MB-CoA to generate C9-CoA.

[0131] Generation of 2MB-CoA The method according to the eighth aspect of the invention comprises overexpressing a heterologous gene encoding a carboxyl coenzyme A (CoA) ligase (CCL) that converts 2-methylbutyrate (2MB) to 2MB-CoA in the presence of exogenously added 2-methylbutyrate. The CCL may be from any plant. In some embodiments, the CCL is QsCCL from Quillaja quinata as set forth in SEQ ID NO: 178, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 178. In another embodiment (not requiring exogenous addition of 2-methylbutyrate), the method of the invention comprises: (i) phosphopantetheinyl transferase (Ppant), and / or (ii) megasynthetases containing an acyl carrier protein (ACP) domain The method further comprises overexpressing a heterologous gene encoding The megasynthetase LovF-TE condenses two units of malonyl-CoA with 2MB-ACP, cleaves 2-methylbutyrate from the ACP domain, and converts 2-methylbutyrate to 2MB-CoA by CCL.

[0132] Ppant may be from Aspergillus nidulans and the megasynthase LovF-TE may be from Aspergillus terreus. In some embodiments, Ppant is AnNpgA as set forth in SEQ ID NO:237 and the megasynthase LovF-TE is AstLovF-TE as set forth in SEQ ID NO:235, or Ppant is a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:237 and the megasynthase LovF-TE is a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:235.

[0133] The eighth aspect of the invention further provides a yeast engineered to produce 2MB-CoA.

[0134] Generation of UDP-arabinofuranose The method according to the ninth aspect of the present invention comprises the steps of: (i) a UDP-Xyl epimerase (UXE) that converts UDP-Xyl to UDP-arabinopyranose (UDP-Arap); and / or (ii) UDP-arabinose mutase (UAM), which converts UDP-Arap to UDP-arabinofuranose (UDP-Araf) The method includes overexpressing a heterologous gene encoding the

[0135] The yeast recombinantly engineered to produce UDP-Xyl may be a yeast according to the fourth aspect of the invention.

[0136] The UXE and UAM may be independently selected from those derived from Arabidopsis and barley. In some embodiments, the UXE is selected from AtUXE as set forth in SEQ ID NO:199, AtUXE2 as set forth in SEQ ID NO:202, HvUXE-1 as set forth in SEQ ID NO:240, HvUXE-2 as set forth in SEQ ID NO:242, AtUGE3 as set forth in SEQ ID NO:205, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:199, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:202, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:240, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:242, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:205.

[0137] In some embodiments, the UAM is selected from AtUAM1 set forth in SEQ ID NO:208, HvUAM set forth in SEQ ID NO:211, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:208, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:211.

[0138] The ninth aspect of the invention further provides a yeast engineered to produce UDP-arabinofuranose.

[0139] Generation of acylated glycosylated QA derivatives As shown in Figure 2A, two acyltransferases sequentially transfer two repeating C9-CoA acyl units to add an 18-carbon pseudodimeric acyl chain to the fucose residue at C28 of the linear tetrasaccharide to generate the acylated glycosylated QA derivative.

[0140] The first step of the method according to the tenth aspect of the invention comprises reacting yeast engineered to produce a glycosylated QA derivative in the presence of exogenously added 2-methylbutyric acid, (i) carboxyl coenzyme A ligase (CCL), which converts 2-methylbutyrate to 2MB-CoA; (ii) a chalcone synthase-like type III polyketide synthase (PKS) that condenses malonyl-CoA with 2MB-CoA to form C9-keto-CoA; (iii) a ketoreductase (KR) that converts C9-keto-CoA to C9-CoA; and / or (iv) an acyltransferase that transfers and attaches a first C9-CoA unit to the glycosylated QA derivative to form an acylated glycosylated QA derivative; The step of overexpressing a heterologous gene encoding

[0141] 2-Methylbutyric acid may, for example, be added directly to the yeast culture medium at the appropriate time.

[0142] In the method according to the tenth aspect of the invention, the glycosylated QA derivative may be QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXA or QA-C3-GGR-C28-FRXA. The yeast engineered to produce QA-C3-GGX-C28-FRX and QA-C3-GGR-C28-FRX may be the yeast of aspect 7c. The yeast engineered to produce QA-C3-GGX-C28-FRXX and QA-C3-GGR-C28-FRXX may be the yeast of aspect 7d. The yeast engineered to produce QA-C3-GGX-C28-FRXA and QA-C3-GGR-C28-FRXA may be a yeast according to embodiment 7e of the invention.

[0143] In the first step of the method according to the tenth aspect of the invention, the CCL may be as described in the method according to the eighth aspect of the invention.

[0144] The chalcone synthase-like type III PKS may be derived from any plant. In some embodiments, the chalcone synthase-like type III PKS is QsChSD as set forth in SEQ ID NO: 181, QsChSE as set forth in SEQ ID NO: 184, both QsChSD as set forth in SEQ ID NO: 181 and QsChSE as set forth in SEQ ID NO: 184, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 181, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 184.

[0145] KR may be derived from any plant. In some embodiments, KR is QsKR11 as set forth in SEQ ID NO: 187, QsKR23 as set forth in SEQ ID NO: 190, both QsKR11 as set forth in SEQ ID NO: 187 and QsKR23 as set forth in SEQ ID NO: 190, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 187, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 190.

[0146] The acyltransferase may be from any plant. In some embodiments, the acyltransferase is QsDMOT9 as set forth in SEQ ID NO: 193, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 187, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 193.

[0147] The tenth aspect of the invention further provides a yeast engineered to produce C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9 or QA-C3-GGR-C28-FRXA-C9 (aspect 10a).

[0148] In the second step of the method according to the tenth aspect of the invention, the acylated glycosylated QA derivative may be C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9 or QA-C3-GGR-C28-FRXA-C9. The yeast engineered to produce C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9 or QA-C3-GGR-C28-FRXA-C9 may be a yeast according to aspect 10a of the invention.

[0149] The third step of the method according to the tenth aspect of the invention further comprises the step of (v) overexpressing a gene encoding a second acyltransferase which attaches a second C9-CoA unit to the acylated glycosylated QA derivative to form a further acylated and glycosylated QA derivative.

[0150] In the third step of the method according to the tenth aspect of the invention, the acylated glycosylated QA derivative may be C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9, or QA-C3-GGR-C28-FRXA-C9. The yeast engineered to produce C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9 and QA-C3-GGR-C28-FRXA-C9 may be a yeast according to embodiment 10a of the invention.

[0151] The acyltransferase may be from any plant. In some embodiments, the acyltransferase is QsDMOT4 as set forth in SEQ ID NO: 196, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 196.

[0152] The tenth aspect of the invention further provides a yeast engineered to produce C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRXA-C18 (aspect 10b).

[0153] The fourth step of the method according to the tenth aspect of the present invention further comprises the step of (vi) overexpressing in the yeast engineered to produce UDP-Araf a heterologous gene encoding an arabinotransferase (ArafT) that transfers UDP-Araf to form a further glycosylated acylated QA derivative by attaching an Araf residue to the acylated glycosylated QA derivative.

[0154] In the fourth step of the method according to the tenth aspect of the invention, the acylated glycosylated QA derivative may be C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRXA-C18. The yeast engineered to produce C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, C3-GGX-C28-FRXA-C18 and QA-C3-GGR-C28-FRXA-C18 may be a yeast according to embodiment 10b of the invention.

[0155] ArafT may be derived from any plant, for example from Quillaja quinata. In some embodiments, ArafT is selected from QsArafT as set forth in SEQ ID NO:229, QsArafT2 as set forth in SEQ ID NO:232, a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:229, and a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:232.

[0156] The tenth aspect of the invention further provides a yeast engineered to produce QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf (aspect 10c).

[0157] In an embodiment in which QsArafT as set forth in SEQ ID NO: 229 is used in the fourth step of the method according to the tenth aspect of the invention, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GGR-C28-FRXA-C18-Xyl or QA-C3-GGX-C28-FRXA-C18-Xyl is further formed. In a tenth aspect of the invention there is further provided a yeast engineered to produce QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GGR-C28-FRXA-C18-Xyl or QA-C3-GGX-C28-FRXA-C18-Xyl (aspect 10d of the invention).

[0158] In the fifth step of the method according to the tenth aspect of the present invention, the method of the present invention comprises the steps of: (vii) phosphopantetheinyl transferase (Ppant), and / or (viii) Megasynthetase LovF-TE containing an acyl carrier protein (ACP) domain The method further comprises overexpressing a heterologous gene encoding The megasynthetase LovF-TE condenses two units of malonyl-CoA with 2MB-ACP, cleaves 2-methylbutyrate from the ACP domain, and converts 2-methylbutyrate to 2MB-CoA by CoA ligase (CCL).

[0159] Ppant may be from Aspergillus nidulans and the megasynthase LovF-TE may be from Aspergillus terreus. In some embodiments, Ppant is AnNpgA as set forth in SEQ ID NO:237 and the megasynthase LovF-TE is AstLovF-TE as set forth in SEQ ID NO:235, or Ppant is a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:237 and the megasynthase LovF-TE is a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:235.

[0160] Sequence identity The "percentage of identity" or "% identity" between a query nucleotide sequence and a subject nucleotide sequence is calculated as a percentage after pairwise global sequence alignment using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or suitable software (e.g., DNASTAR Lasergene or GenePAST / KERR), and the "identity" value is calculated over the entire length of the query sequence using a suitable algorithm (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN or GenePAST / KERR) or suitable software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign). Importantly, the query nucleotide sequence may be any of the nucleotide sequences disclosed herein, and in particular any of the nucleotide sequences disclosed in one or more claims.

[0161] The "percentage of identity" or "% identity" between a query amino acid sequence and a subject amino acid sequence is calculated as a percentage after pairwise global sequence alignment using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or suitable software (e.g., DNASTAR Lasergene or GenePAST / KERR), and the "identity" value is calculated over the entire length of the query sequence using a suitable algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN or GenePAST / KERR) or suitable software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign). Importantly, the query amino acid sequence may be any of the amino acid sequences disclosed herein, and in particular any of the amino acid sequences disclosed in one or more claims.

[0162] The query sequence may have 100% identity with the subject sequence or may contain amino acid or nucleotide changes, represented by a specific integer value, that result in a percentage of identity less than 100% compared to the subject sequence. For example, the query sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the subject sequence. In the case of nucleotide sequences, such changes include the deletion, substitution or insertion of at least one nucleotide residue, which may be at the 5' or 3' position of the query sequence, anywhere in between the two ends, interspersed among the nucleotide residues of the query sequence, or in one or more contiguous groups within the query sequence. In the case of amino acid sequences, such changes include deletions, substitutions (including conservative and non-conservative substitutions), or insertions of at least one amino acid residue, which may be at the amino-terminal or carboxy-terminal positions of the query sequence, or anywhere in between the termini, and which may be interspersed individually among the amino acid residues of the query sequence, or may be present in one or more contiguous groups within the query sequence.

[0163] With respect to the enzymes and / or proteins used in the methods of the invention and for which sequence identity is defined, such enzymes and / or proteins typically retain the function and activity of these enzymes and / or proteins, which may be assessed as described in the Examples.

[0164] Yeast recombination The yeast may be modified in the method of the invention using conventional methods (see, for example, US Patent Publication No. 8,828,684 (B2), the contents of which are incorporated herein by reference). The heterologous gene may be expressed under the control of a constitutive or inducible promoter, for example, under the control of a galactose-inducible promoter. The gene may be expressed by integration into the genome of the particular yeast strain (at one locus or at multiple different loci) or via plasmid expression. When using integration into the genome, one or more copies of the gene to be overexpressed may be integrated, for example, 1-10 copies, 2-8 copies, 3-7 copies of the gene may be integrated. In some embodiments, one or more genes involved in the biosynthesis of QS-21 are integrated into the yeast genome. General yeast culture conditions are known to those of skill in the art. The recombinant yeast may be cultured for several days, for example, 1-7 days, 2-6 days, 4-5 days, or 3 days. It is within the skill of the art to determine the optimal culture time depending on the metabolic product to be produced. When using an inducible promoter such as the gal promoter, it is also within the skill of the art to determine optimal induction times. After an appropriate period of cultivation and / or induction, the desired metabolite (e.g., a sugar or QA derivative of the invention) may be recovered from the yeast culture by methods known in the art, including extraction using non-aqueous polar solvents, extraction using acidic or basic media, recovery by resin adsorption, extraction by mechanical disruption of plant cells using ball milling, ultrasonic disruption, and the like. In some embodiments, the yeast is Saccharomyces cerevisiae.

[0165] Adjuvants The QA derivative of the present invention can be used as an adjuvant, alone or in combination.The QA derivative of the present invention can be further combined with another immunostimulant, and in particular with a TLR4 agonist.In some embodiments, the QA derivative of the present invention is formulated in liposome in combination with a TLR4 agonist.

[0166] The TLR4 agonist may be 3D-MPL, in particular a lipopolysaccharide TLR4 agonist such as lipid A derivative, in particular monophosphoryl lipid A, for example 3-O-deacylated monophosphoryl lipid A (3D-MPL). 3D-MPL is sold by GlaxoSmithKline Biologicals NA under the name "MPL". See, for example, U.S. Pat. No. 4,436,727, U.S. Pat. No. 4,877,611, U.S. Pat. No. 4,866,034 and U.S. Pat. No. 4,912,094. 3D-MPL may be produced according to the method described in British Patent Publication 2 220 211 (A). Chemically, 3D-MPL is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.

[0167] The adjuvants of the invention may be formulated in a suitable carrier, such as an emulsion (eg, an oil-in-water emulsion) or liposome, as described below.

[0168] Liposomes The term "liposome" is known in the art and defines a general classification of vesicles that contain one or more lipid bilayers surrounding an aqueous space. Liposomes therefore consist of one or more bilayers of lipids and / or phospholipids and can contain other molecules, such as proteins or carbohydrates, within their structure. Liposomes have both lipid and aqueous phases and therefore can encapsulate or entrap water-soluble materials, fat-soluble materials, and / or amphiphilic compounds. Methods for making such liposomes are described in WO13 / 041572.

[0169] The size of the liposomes may range from 30 nm to several microns, depending on the phospholipid composition and the method used for their preparation.

[0170] The size of the liposomes is in the range of 50 nm to 200 nm, particularly 60 nm to 180 nm, for example 70 nm to 165 nm. Optimally, the liposomes have a diameter of 100 nm so that they can be easily sterilized by filtration, and are stable.

[0171] The structural integrity of the liposomes may be assessed by dynamic light scattering (DLS) to measure the size (Z-average diameter, Zav) and polydispersity of the liposomes, or by electron microscopy to analyze the structure of the liposomes. The average particle size may be between 95 nm and 120 nm, and / or the polydispersity (Pdl) index may be 0.3 or less (e.g., 0.2 or less).

[0172] [Table 1] TIFF2025501616000003.tif210165 TIFF2025501616000004.tif212165 TIFF2025501616000005.tif216164 TIFF2025501616000006.tif218164 TIFF2025501616000007.tif245163 TIFF2025501616000008.tif242163 TIFF2025501616000009.tif231163 TIFF2025501616000010.tif220164 TIFF2025501616000011.tif216164 TIFF2025501616000012.tif190163 TIFF2025501616000013.tif196164 TIFF2025501616000014.tif196164 TIFF2025501616000015.tif182163 TIFF2025501616000016.tif118164 TIFF2025501616000017.tif199163 TIFF2025501616000018.tif204163 TIFF2025501616000019.tif207163 TIFF2025501616000020.tif189164 TIFF2025501616000021.tif137164 TIFF2025501616000022.tif120164 TIFF2025501616000023.tif232163

[0173] Throughout this specification, including the claims, unless the context indicates otherwise, the term "comprises" and variations thereof, such as "comprising," are to be construed to include a component (e.g., an integer) or components (e.g., integers) described herein, without necessarily excluding other components (e.g., integers). Thus, a composition "comprising" X may consist exclusively of X, or may include additional components (e.g., X+Y).

[0174] The term "substantially" does not exclude the term "completely", e.g. a composition that is "substantially free" of Y may mean a composition that is completely free of Y. If desired, the term "substantially" may be omitted from the definition of the invention.

[0175] The term "about" or "approximately" used in reference to a numerical value x refers to options, such as meaning ±10% of a numerical value x, such as meaning ±5% of a numerical value x, and specifically referring to that numerical value.

[0176] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0177] As used herein, ng means nanograms, ug or μg means micrograms, mg means milligrams, mL or ml means milliliters, and mM means millimolar. Similar terms such as μm are to be interpreted similarly.

[0178] Unless otherwise specified, a method that includes a step of mixing two or more components does not require that the components be mixed in a particular order. Thus, the two or more components can be mixed in any order. For example, if three components are present, two components can be mixed, and then the resulting mixture can be mixed with a third component, etc.

[0179] The invention is further described by reference to the following sections.

[0180] 1. A method for producing quilacic acid (QA) in yeast, comprising: In yeast engineered to produce β-amyrin, (i) cytochrome P450 C16 oxidase, which oxidizes the carbon at the C16 position of β-amyrin to generate a hydroxyl group; (ii) cytochrome P450 C23 oxidase, which oxidizes the carbon at C23 of β-amyrin to generate an aldehyde group; (iii) cytochrome P450 C28 oxidase, which oxidizes the carbon at C28 of β-amyrin to generate a carboxyl group; and / or (iv) Cytochrome P450 reductase (CPR) acting as a redox partner overexpressing a heterologous gene encoding The method, wherein the C16 oxidase, the C23 oxidase, the C28 oxidase and the CPR are derived from a plant.

[0181] Item 2. The method of item 1, wherein the C16 oxidase, the C23 oxidase and the C28 oxidase are independently selected from those derived from Artemisia annua (Aa), Arabidopsis thaliana (At), Glycyrrhiza glabra (Gg), Medicago truncatula (Mt), Quillaja saponaria (Qs), Saponaria vaccaria (Sv), Centella asiatica (Ca), Bupleurum falcatum (Bf) and Maesa lanceolata (Ml).

[0182] Item 3. The method of item 1 or 2, wherein the C16 oxidase, the C23 oxidase, and the C28 oxidase are independently selected from those derived from Medicago truncatula (Mt), Bupleurum falcatum (Bf), Quillaja saponaria (Qs), and Saponaria vaccaria (Sv).

[0183] Clause 4. The method of clause 3, wherein the C16 oxidase is selected from QsC16 as set forth in SEQ ID NO:20, QsC28C16 as set forth in SEQ ID NO:23, and SvC16 as set forth in SEQ ID NO:26.

[0184] Clause 5. The method of clause 4, wherein the QsC16 is encoded by the nucleotide sequence of SEQ ID NO:21, the QsC28C16 is encoded by the nucleotide sequence of SEQ ID NO:24, and the SvC16 is encoded by the nucleotide sequence of SEQ ID NO:27.

[0185] Item 6. The method according to any one of items 1 to 5, wherein the C23 oxidase is selected from MtC23 oxidase shown in SEQ ID NO: 38, QsC23 shown in SEQ ID NO: 29, SvC23-1 shown in SEQ ID NO: 32, and SvC23-2 shown in SEQ ID NO: 35.

[0186] Clause 7. The method of clause 6, wherein the MtC23 is encoded by the nucleotide sequence of SEQ ID NO:39, the QsC23 is encoded by the nucleotide sequence of SEQ ID NO:30, the SvC23-1 is encoded by the nucleotide sequence of SEQ ID NO:33, and the SvC23-2 is encoded by the nucleotide sequence of SEQ ID NO:36.

[0187] Item 8. The method according to any one of items 1 to 7, wherein the C28 oxidase is selected from MtC28 shown in SEQ ID NO: 46, QsC28 shown in SEQ ID NO: 41, and SvC28 shown in SEQ ID NO: 44.

[0188] Clause 9. The method of clause 8, wherein said MtC28 is encoded by the nucleotide sequence of SEQ ID NO:47, said QsC28 is encoded by the nucleotide sequence of SEQ ID NO:42, and said SvC28 is encoded by the nucleotide sequence of SEQ ID NO:45.

[0189] Item 10. The method according to any one of items 4 to 9, wherein the C16 oxidase is SvC16 as shown in SEQ ID NO: 26, the C23 oxidase is SvC23-1 as shown in SEQ ID NO: 32 or SvC23-2 as shown in SEQ ID NO: 35, and the C28 oxidase is SvC28 as shown in SEQ ID NO: 44.

[0190] Clause 11. The method of clause 10, wherein the SvC16 is encoded by the nucleotide sequence of SEQ ID NO:27, the SvC23-1 is encoded by the nucleotide sequence of SEQ ID NO:33, the SvC23-2 is encoded by the nucleotide sequence of SEQ ID NO:36, and the SvC28 is encoded by the nucleotide sequence of SEQ ID NO:45.

[0191] Item 12. The method according to any one of items 4 to 9, wherein the C16 oxidase is selected from QsC16 shown in SEQ ID NO: 20 and QsC28C16 shown in SEQ ID NO: 23, the C23 oxidase is QsC23 shown in SEQ ID NO: 29, and the C28 oxidase is QsC28 shown in SEQ ID NO: 41.

[0192] Clause 13. The method of clause 12, wherein the QsC16 is encoded by the nucleotide sequence of SEQ ID NO:21, the QsC28C16 is encoded by the nucleotide sequence of SEQ ID NO:24, the QsC23 is encoded by the nucleotide sequence of SEQ ID NO:30, and the QsC28 is encoded by the nucleotide sequence of SEQ ID NO:42.

[0193] Item 14. The method according to any one of items 4 to 9, wherein the C16 oxidase is QsC28C16 as shown in SEQ ID NO: 23, the C23 oxidase is QsC23 as shown in SEQ ID NO: 29, and the C28 oxidase is QsC28 as shown in SEQ ID NO: 41.

[0194] Clause 15. The method of clause 14, wherein the QsC28C16 is encoded by the nucleotide sequence of SEQ ID NO:24, the QsC23 is encoded by the nucleotide sequence of SEQ ID NO:30, and the QsC28 is encoded by the nucleotide sequence of SEQ ID NO:42.

[0195] Clause 16. The method of any one of clauses 1 to 15, wherein the CPR is selected from those derived from A. thaliana (At) and Lotus japonicus (Lj).

[0196] Clause 17. The method of clause 16, wherein the CPR is selected from AtATR1 as set forth in SEQ ID NO:49, and LjCPR as set forth in SEQ ID NO:52.

[0197] Clause 18. The method of clause 17, wherein the CPR is AtATR1 as set forth in SEQ ID NO:49.

[0198] Clause 19. The method of clause 17 or clause 18, wherein said AtATR1 is encoded by the nucleotide sequence of SEQ ID NO:50 and said LjCPR is encoded by the nucleotide sequence of SEQ ID NO:53.

[0199] Item 20. The method according to any one of items 1 to 19, wherein the yeast further overexpresses (v) a heterologous gene encoding cytochrome b5.

[0200] Clause 21. The method of clause 20, wherein the cytochrome b5 is selected from those derived from A. thaliana (At), Q. saponaria (Qs) and S. vaccaria (Sv).

[0201] Clause 22. The method of clause 21, wherein the cytochrome b5 is selected from Atb5 as set forth in SEQ ID NO:58, Qsb5 as set forth in SEQ ID NO:55 and Svb5 as set forth in SEQ ID NO:61.

[0202] Clause 23. The method of clause 21 or clause 22, wherein the cytochrome b5 is Qsb5 as set forth in SEQ ID NO:55.

[0203] Clause 24. The method of clause 21 or clause 22, wherein the cytochrome b5 is Svb5 as set forth in SEQ ID NO:61.

[0204] Clause 25. The method according to any one of clauses 22 to 24, wherein the Atb5 is encoded by the nucleotide sequence of SEQ ID NO: 59, the Qsb5 is encoded by the nucleotide sequence of SEQ ID NO: 56, and the Svb5 is encoded by the nucleotide sequence of SEQ ID NO: 62.

[0205] Item 26. The method of any one of items 1 to 25, wherein the yeast further overexpresses (vi) a heterologous gene encoding a scaffold protein that physically interacts with one or more of the C16 oxidase, the C23 oxidase, the C28 oxidase, and the CPR.

[0206] Clause 27. The method of clause 26, wherein the scaffold protein is a membrane steroid binding protein (MSBP).

[0207] Clause 28. The method of clause 27, wherein said MSBP is selected from those derived from A. thaliana (At), Q. saponaria (Qs) and S. vaccaria (Sv).

[0208] Clause 29. The method of clause 27 or clause 28, wherein said MSBP is selected from AtMSBP1 as set forth in SEQ ID NO:63, and AtMSBP2 as set forth in SEQ ID NO:65.

[0209] Clause 30. The method of clause 27 or clause 28, wherein said MSBP is selected from QsMSBP1 as set forth in SEQ ID NO: 73, SvMSBP1 as set forth in SEQ ID NO: 67, and SvMSBP2 as set forth in SEQ ID NO: 70.

[0210] Clause 31. The method of clause 27, clause 28 or clause 30, wherein said MSBP is SvMSBP1 as set forth in SEQ ID NO:67.

[0211] Clause 32: The method according to any one of clauses 29 to 31, wherein the AtMSBP1 is encoded by the nucleotide sequence of SEQ ID NO: 64, the AtMSBP2 is encoded by the nucleotide sequence of SEQ ID NO: 66, the QsMSBP1 is encoded by the nucleotide sequence of SEQ ID NO: 74, the SvMSBP1 is encoded by the nucleotide sequence of SEQ ID NO: 68, and the SvMSBP2 is encoded by the nucleotide sequence of SEQ ID NO: 71.

[0212] Item 33. The method according to any one of items 1 to 32, wherein the yeast recombinantly engineered to produce β-amyrin overexpresses a β-amyrin synthase (BAS) selected from those derived from Artemisia annua (Aa), Arabidopsis thaliana (A. thaliana) (At), G. glabra (Gg), G. vaccaria (Gv), Saponaria vaccaria (Sv), and Q. saponaria (Qs).

[0213] Clause 34. The method of clause 33, wherein the BAS is selected from AaBAS as set forth in SEQ ID NO:1, AtBAS as set forth in SEQ ID NO:4, GgBAS as set forth in SEQ ID NO:7, GvBAS as set forth in SEQ ID NO:10, QsBAS as set forth in SEQ ID NO:15, and SvBAS as set forth in SEQ ID NO:13.

[0214] Clause 35. The method of clause 33 or clause 34, wherein the BAS is GvBAS as set forth in SEQ ID NO:10.

[0215] Clause 36. The method of clause 34 or clause 35, wherein the AaBAS is encoded by the nucleotide sequence of SEQ ID NO:2, the AtBAS is encoded by the nucleotide sequence of SEQ ID NO:5, the GgBAS is encoded by the nucleotide sequence of SEQ ID NO:8, the GvBAS is encoded by the nucleotide sequence of SEQ ID NO:11, the QsBAS is encoded by the nucleotide sequence of SEQ ID NO:16, and the SvBAS is encoded by the nucleotide sequence of SEQ ID NO:14.

[0216] Clause 37. The method according to any one of clauses 1 to 9, 12 to 23, 25 to 28 and 30 to 36, wherein the C16 oxidase is QsC28C16, the C23 oxidase is QsC23, the C28 oxidase is QsC28, the CPR is AtATR1, the MSBP is SvMSBP1, the cytochrome b5 is Qsb5 and the BAS is GvBAS.

[0217] Clause 38. The method of clause 37, wherein the QsC28C16 is encoded by the nucleotide sequence of SEQ ID NO:24, the QsC23 is encoded by the nucleotide sequence of SEQ ID NO:30, the QsC28 is encoded by the nucleotide sequence of SEQ ID NO:42, the AtATR1 is encoded by the nucleotide sequence of SEQ ID NO:24, the SvMSBP1 is encoded by the nucleotide sequence of SEQ ID NO:50, the Qsb5 is encoded by the nucleotide sequence of SEQ ID NO:56, and the GvBAS is encoded by the nucleotide sequence of SEQ ID NO:11.

[0218] Item 39. A yeast recombinantly produced to produce quillic acid (QA) by the method according to any one of items 1 to 38.

[0219] Item 40. The yeast of item 39, producing at least 60 mg / L of QA.

[0220] 41. A method for producing UDP-glucuronic acid (UDP-GlcA) in yeast, comprising: A method comprising overexpressing a heterologous gene encoding UDP-glucose dehydrogenase (UGD), which converts UDP-glucose (UDP-Glc) to UDP-GlcA.

[0221] Clause 42. The method of clause 41, wherein said UGD is derived from Arabidopsis thaliana (A. thaliana) (At).

[0222] 43. The UGD is selected from the group consisting of AtUGD as set forth in SEQ ID NO: 84 and AtUGD as set forth in SEQ ID NO: 108. A101L 40. The method of claim 39, wherein the method is selected from the group consisting of:

[0223] 44. The UGD according to claim 1, wherein the UGD is AtUGD as set forth in SEQ ID NO: 108. A101L 44. The method according to any one of items 41 to 43, wherein

[0224] Clause 45. The AtUGD is encoded by the nucleotide sequence of SEQ ID NO: 85, A101L 45. The method of claim 43 or 44, wherein said vector is encoded by the nucleotide sequence of SEQ ID NO:109.

[0225] Item 46. A yeast recombinantly produced to produce UDP-GlcA by the method according to any one of items 41 to 45.

[0226] 47. A method for producing UDP-rhamnose (UDP-Rha) in yeast, comprising the steps of: A method comprising overexpressing a heterologous gene encoding UDP-rhamnose synthase, which converts UDP-glucose (UDP-Glc) to UDP-Rha.

[0227] Clause 48. The method of clause 47, wherein the UDP-rhamnose synthase is derived from A. thaliana (At).

[0228] Clause 49. The method according to clause 48, wherein said UDP-rhamnose synthase is AtRHM2 as set forth in SEQ ID NO:102.

[0229] Clause 50. The method of clause 49, wherein said AtRHM2 is encoded by the nucleotide sequence of SEQ ID NO:103.

[0230] Paragraph 51. A yeast recombinantly produced by the method according to any one of paragraphs 47 to 50, wherein the yeast is capable of producing UDP-Rha.

[0231] 52. A method for producing UDP-xylose (UDP-Xyl) in yeast, comprising: (i) UDP-glucose dehydrogenase (UGD), which converts UDP-Glc to UDP-GlcA; and / or (ii) UDP-xylose synthase (UXS), which converts UDP-GlcA to UDP-xylose. The method comprises overexpressing a heterologous gene encoding the

[0232] Clause 53. The method of clause 52, wherein said UGD and said UXS are independently selected from those derived from A. thaliana (At) and Q. saponaria (Qs).

[0233] 54. The UGD is selected from the group consisting of AtUGD as set forth in SEQ ID NO: 84 and AtUGD as set forth in SEQ ID NO: 108. A101L 54. The method of claim 53, wherein the method is selected from the group consisting of

[0234] Clause 55. The method of clause 52, wherein the UGD is selected from those derived from Synechococcus (Syn), Homo sapiens (Hs), Paramoeba atlantica (Patl), Bacillus cytotoxicus (Bcyt), Corallococcus macrosporus (Myxfulv), and Pyrococcus furiosus (Pfu).

[0235] 56. The UGD is selected from the group consisting of SynUGD as shown in SEQ ID NO: 154 and HsUGD as shown in SEQ ID NO: 157. A104L 56. The method of claim 55, wherein the UGD is selected from the group consisting of PatlUGD as set forth in SEQ ID NO: 110, BcytUGD as set forth in SEQ ID NO: 160, MyxfulvUGD as set forth in SEQ ID NO: 163, and PfuUGD as set forth in SEQ ID NO: 166.

[0236] 57. The UGD according to claim 1, wherein the UGD is AtUGD as set forth in SEQ ID NO: 108. A101L 55. The method according to any one of items 52 to 54, wherein

[0237] Clause 58. The AtUGD is encoded by the nucleotide sequence of SEQ ID NO: 85. A101L is encoded by the nucleotide sequence of SEQ ID NO: 109, the SynUGD is encoded by the nucleotide sequence of SEQ ID NO: 155, and the HsUGD A104LThe method according to any one of items 54 to 57, wherein the PatlUGD is encoded by the nucleotide sequence of SEQ ID NO: 158, the PatlUGD is encoded by the nucleotide sequence of SEQ ID NO: 111, the BcytUGD is encoded by the nucleotide sequence of SEQ ID NO: 161, the MyxfulvUGD is encoded by the nucleotide sequence of SEQ ID NO: 164, and the PfuUGD is encoded by the nucleotide sequence of SEQ ID NO: 167.

[0238] Clause 59. The method according to any one of clauses 52 to 58, wherein said UXS is selected from AtUXS as set forth in SEQ ID NO:105, and QsAXS as set forth in SEQ ID NO:113.

[0239] 60. The UGD according to claim 60, wherein the UGD is AtUGD as set forth in SEQ ID NO: 108. A101L 61. The method of claim 60, wherein the UXS is AtUXS as set forth in SEQ ID NO: 105.

[0240] Clause 61. The AtUXS is encoded by the nucleotide sequence of SEQ ID NO: 106, the QsAXS is encoded by the nucleotide sequence of SEQ ID NO: 114, and the AtUGD A101L 61. The method of claim 59 or 60, wherein said vector is encoded by the nucleotide sequence of SEQ ID NO:109.

[0241] Paragraph 62: A yeast recombinantly produced to produce UDP-Xyl by the method according to any one of paragraphs 52 to 61.

[0242] 63. A method for producing a C3-glycosylated QA derivative in yeast, comprising the steps of: the C3-glycosylated QA derivative is QA-C3-GlcA, The method comprises the steps of: in a yeast recombinantly engineered to produce QA and UDP-GlcA, (i) UDP-GlcA transferase (GlcAT), which transfers UDP-GlcA and attaches a GlcA residue to the C3 position of QA, forming QA-C3-GlcA. The method comprises overexpressing a heterologous gene encoding the

[0243] Clause 64. The method of clause 63, wherein the GlcAT is selected from those derived from Q. saponaria (Qs) and S. vaccaria (Sv).

[0244] Clause 65. The method of clause 63 or clause 64, wherein the GlcAT is derived from Q. saponaria.

[0245] Clause 66. The method of clause 65, wherein said GlcAT is selected from QsCslG1 as set forth in SEQ ID NO:78, and QsCslG2 as set forth in SEQ ID NO:81.

[0246] Clause 67. The method of clause 66, wherein said GlcAT is QsCslG2 as set forth in SEQ ID NO:81.

[0247] Clause 68. The method of clause 64, wherein the GlcAT is derived from Saponaria vaccaria.

[0248] Clause 69. The method of clause 68, wherein said GlcAT is SvCslG as set forth in SEQ ID NO:76.

[0249] Clause 70. The method of any one of clauses 66, 67, 68 or 69, wherein said QsCslG1 is encoded by the nucleotide sequence of SEQ ID NO:79, said QsCslG2 is encoded by the nucleotide sequence of SEQ ID NO:82, and said SvCslG is encoded by the nucleotide sequence of SEQ ID NO:77.

[0250] Clause 71. The method according to any one of clauses 63 to 70, wherein said yeast recombinantly engineered to produce QA is a yeast according to clause 39.

[0251] Clause 72. The method of clause 71, wherein the yeast recombinantly produced UDP-GlcA is a yeast according to clause 46.

[0252] Clause 73. A yeast recombinantly produced to produce QA-C3-GlcA by the method according to any one of clauses 63 to 72.

[0253] 74. The method of claim 73, wherein the C3-glycosylated QA derivative is QA-C3-GlcA-Gal, and the overexpression is (ii) UDP-galactosetransferase (GalT), which transfers UDP-Gal and attaches a Gal residue to QA-C3-GlcA to form QA-C3-GlcA-Gal; 73. The method of any one of items 63 to 72, further comprising overexpressing a heterologous gene encoding the

[0254] Clause 75. The method of clause 74, wherein said GalT is selected from those derived from Q. saponaria (Qs) and S. vaccaria (Sv).

[0255] Clause 76. The method of clause 75, wherein the GalT is derived from Q. saponaria (Qs).

[0256] Clause 77. The method according to any one of clauses 70 to 76, wherein said GalT is QsGalT as shown in SEQ ID NO:116.

[0257] Clause 78. The method of clause 74, wherein said GalT is derived from Saponaria vaccaria.

[0258] Clause 79. The method of clause 78, wherein said GalT is SvGalT as set forth in SEQ ID NO:98.

[0259] Clause 80. The method of clause 77 or clause 79, wherein said QsGalT is encoded by the nucleotide sequence of SEQ ID NO:117 and said SvGalT is encoded by the nucleotide sequence of SEQ ID NO:99.

[0260] Clause 81. A yeast recombinantly produced by the method according to any one of clauses 74 to 80, wherein the yeast is capable of producing QA-C3-GlcA-Gal.

[0261] Clause 82. The C3-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha, the yeast is further engineered to produce UDP-Rha, and the overexpression (iii) UDP-rhamnose transferase (RhaT), which transfers UDP-Rha and attaches the Rha residue to QA-C3-GlcA-Gal to form QA-C3-GlcA-Gal-Rha. 81. The method of any one of paragraphs 74 to 80, further comprising overexpressing a heterologous gene encoding the

[0262] Clause 83. The method of clause 82, wherein the RhaT is derived from Q. saponaria (Qs).

[0263] Clause 84. The method of clause 83, wherein said RhaT is QsRhaT as set forth in SEQ ID NO:119.

[0264] Clause 85. The method of clause 84, wherein said QsRhaT is encoded by the nucleotide sequence of SEQ ID NO:120.

[0265] Clause 86. The method according to any one of clauses 82 to 86, wherein the yeast recombinantly produced UDP-Rha is a yeast according to clause 51.

[0266] Clause 87. A yeast recombinantly produced by the method according to any one of clauses 82 to 86, wherein the yeast is capable of producing QA-C3-GlcA-Gal-Rha.

[0267] Clause 88. The C3-glycosylated QA derivative is QA-C3-GlcA-Gal-Xyl, the yeast is further engineered to produce UDP-Xyl, and the overexpression (iv) UDP-xylose transferase (XylT), which transfers UDP-xylose to attach a Xyl residue to QA-C3-GlcA-Gal to form QA-C3-GlcA-Gal-Xyl. 81. The method of any one of paragraphs 74 to 80, further comprising overexpressing a heterologous gene encoding the

[0268] Clause 89. The method of clause 88, wherein the XylTt is selected from those derived from Q. saponaria (Qs) or S. vaccaria (Sv).

[0269] Clause 90. The method of clause 89, wherein said XylT is selected from QsC3XylT as set forth in SEQ ID NO:122, and SvC3XylT as set forth in SEQ ID NO:100.

[0270] Clause 91. The method of clause 90, wherein the QsC3XylT is encoded by the nucleotide sequence of SEQ ID NO: 123 and the SvC3XylT is encoded by the nucleotide sequence of SEQ ID NO: 101, and the yeast recombinantly engineered to produce UDP-Xyl is the yeast of clause 62.

[0271] Clause 92. A yeast recombinantly produced by the method according to any one of clauses 88 to 91, wherein the yeast is capable of producing QA-C3-GlcA-Gal-Xyl.

[0272] Clause 93: The overexpression is in the presence of exogenously added glucuronic acid, (v) glucuronokinase (GlcAK), which converts free glucuronic acid to GlcA-1-phosphate; and / or (vi) UDP-sugar pyrophosphorylase (USP), which converts GlcA-1-phosphate to UDP-GlcA 92. The method of any one of items 88 to 91, further comprising overexpressing a heterologous gene encoding the

[0273] Clause 94. The method of clause 93, wherein said GlcAK and said USP are derived from A. thaliana (At).

[0274] Clause 95. The method of clause 94, wherein said GlcAK is AtGlcAK as set forth in SEQ ID NO: 169 and said USP is AtUSP as set forth in SEQ ID NO: 223.

[0275] Clause 96. The method of clause 95, wherein said AtGlcAK is encoded by the nucleotide sequence of SEQ ID NO: 170 and said AtUSP is encoded by the nucleotide sequence of SEQ ID NO: 224.

[0276] Clause 97. The method of any one of clauses 93 to 96, wherein said overexpression further comprises overexpressing a heterologous gene encoding (vi) myo-inositol oxygenase (MIOX) in the presence of exogenously added myo-inositol.

[0277] Clause 98. The method of clause 97, wherein the MIOX is derived from Thermothelomyces thermophilus (Tt).

[0278] Clause 99. The method of clause 98, wherein said MIOX is TtMIOX as set forth in SEQ ID NO:173.

[0279] Clause 100. The method of clause 99, wherein said TtMIOX is encoded by the nucleotide sequence of SEQ ID NO:174.

[0280] Clause 101. A yeast recombinantly produced by the method according to any one of clauses 93 to 100, wherein the yeast is capable of producing QA-C3-GlcA-Gal-Xyl.

[0281] Item 102. A method for producing UDP-fucose (UDP-Fuc) in yeast, comprising: (i) UDP-glucose-4,6-dehydratase (UG46DH), which converts UDP-Glc to UDP-4-keto-6-deoxyglucose, and / or (ii) 4-ketoreductase that converts UDP-4-keto-6-deoxyglucose to UDP-D-Fuc The method comprises overexpressing a heterologous gene encoding the

[0282] Clause 103. The method of clause 102, wherein said UG46DH is derived from Saponaria vaccaria (Sv).

[0283] Clause 104. The method of clause 103, wherein said UG46DH is SvUG46DH as set forth in SEQ ID NO:87.

[0284] Clause 105. The method of clause 104, wherein said SvUG46DH is encoded by the nucleotide sequence of SEQ ID NO:88.

[0285] Clause 106. The method of any one of clauses 102 to 105, wherein the 4-ketoreductase is selected from those derived from Q. saponaria (Qs) and S. vaccaria (Sv).

[0286] Clause 107. The method of clause 106, wherein the 4-ketoreductase is selected from svNMD as set forth in SEQ ID NO:90, and QsFucSyn as set forth in SEQ ID NO:175.

[0287] Clause 108. The method of clause 107, wherein said svNMD is encoded by the nucleotide sequence of SEQ ID NO:91 and said QsFucSyn is encoded by the nucleotide sequence of SEQ ID NO:176.

[0288] Clause 109. A yeast recombinantly produced to produce UDP-fucose by the method according to any one of clauses 102 to 108.

[0289] 110. A method for producing a C28-glycosylated QA derivative in yeast, comprising: the C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc or QA-C3-GlcA-Gal-Xyl-C28-Fuc, and the method comprises the steps of: (i) UDP-fucose transferase (FucT), which transfers UDP-Fuc to attach a Fuc residue to the C28 position of QA, forming QA-C3-GlcA-Gal-Rha-C28-Fuc or QA-C3-GlcA-Gal-Xyl-C28-Fuc. The method comprises overexpressing a heterologous gene encoding the

[0290] Clause 111. The method of clause 110, wherein the FucT is selected from those derived from Q. saponaria (Qs) and S. vaccaria (Sv).

[0291] Clause 112. The method of clause 111, wherein said FucT is selected from QsFucT as set forth in SEQ ID NO:93, and SvFucT as set forth in SEQ ID NO:96.

[0292] Clause 113. The method of clause 112, wherein said QsFucT is encoded by the nucleotide sequence of SEQ ID NO:94 and said SvFucT is encoded by the nucleotide sequence of SEQ ID NO:97.

[0293] Clause 114. The method according to any one of clauses 110 to 113, wherein the yeast recombinantly produced by the recombinant yeast is the yeast according to clause 87, and the yeast recombinantly produced by the recombinant yeast is the yeast according to clause 109.

[0294] Clause 115: The method according to any one of clauses 110 to 113, wherein the yeast recombinantly produced by the recombinant yeast is the yeast according to clause 101, and the yeast recombinantly produced by the recombinant yeast is the yeast according to clause 109.

[0295] Clause 116. A yeast recombinantly produced by the method according to any one of clauses 110 to 114, wherein the yeast is capable of producing QA-C3-GlcA-Gal-Rha-C28-Fuc.

[0296] Clause 117. A yeast recombinantly produced by the method according to any one of clauses 110 to 113 and 115, which produces QA-C3-GlcA-Gal-Xyl-C28-Fuc.

[0297] Clause 118. The C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha, and the overexpression is (ii) UDP-rhamnose transferase (RhaT), which transfers UDP-Rha to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha by linking the Rha residue to QA-C3-GlcA-Gal-Rha-C28-Fuc or QA-C3-GlcA-Gal-Xyl-C28-Fuc. 116. The method of any one of paragraphs 110 to 115, further comprising overexpressing a heterologous gene encoding the

[0298] Clause 119. The method of clause 118, wherein the RhaT is derived from Q. saponaria.

[0299] Clause 120. The method of clause 119, wherein said RhaT is QsRhaT as set forth in SEQ ID NO:119.

[0300] Clause 121. The method of clause 120, wherein said QsRhaT is encoded by the nucleotide sequence of SEQ ID NO:120.

[0301] Clause 122. A yeast recombinantly produced by the method according to any one of clauses 118 to 121, comprising the step of:

[0302] Clause 123. The C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl, and the overexpression is (iii) UDP-xylosetransferase (XylT), which transfers UDP-Xyl and attaches a Xyl residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha to form GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl, respectively. 122. The method of any one of items 118 to 121, further comprising overexpressing a heterologous gene encoding the

[0303] Clause 124. The method of clause 123, wherein said XylT is selected from those derived from Q. saponaria (Qs) and S. vaccaria (Sv).

[0304] Clause 125. The method of clause 124, wherein said XylT is QsC28XylT3 as set forth in SEQ ID NO:125.

[0305] Clause 126. The method of clause 125, wherein said QsC28XylT3 is encoded by the nucleotide sequence of SEQ ID NO:126.

[0306] Clause 127. A yeast recombinantly produced by the method according to any one of clauses 123 to 126, comprising QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl.

[0307] Clause 128. The C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, and the overexpression is (iv) UDP-xylosetransferase (XylT), which transfers UDP-Xyl and attaches a Xyl residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, respectively. 127. The method of any one of paragraphs 123 to 126, further comprising overexpressing a heterologous gene encoding the

[0308] Clause 129. The method of clause 128, wherein said XylT is selected from those derived from Q. saponaria (Qs) and S. vaccaria (Sv).

[0309] Clause 130. The method of clause 129, wherein said XylT is QsC28XylT4 as set forth in SEQ ID NO:128.

[0310] Clause 131. The method of clause 130, wherein said QsC28XylT4 is encoded by the nucleotide sequence of SEQ ID NO:129.

[0311] Clause 132. The method of clause 128 or clause 129, wherein said QsC28XylT4 comprises an amino acid deletion of 3 to 20 amino acid residues at the N-terminus.

[0312] Clause 133. The method of clause 132, wherein said XylT is selected from QsC28XylT4-3aa as set forth in SEQ ID NO: 131, QsC28XylT4-6aa as set forth in SEQ ID NO: 134, QsC28XylT4-9aa as set forth in SEQ ID NO: 137, and QsC28XylT4-12aa as set forth in SEQ ID NO: 140.

[0313] Clause 134. The method of clause 133, wherein said QsC28XylT4-3aa is encoded by the nucleotide sequence of SEQ ID NO: 132, said QsC28XylT4-6aa is encoded by the nucleotide sequence of SEQ ID NO: 135, said QsC28XylT4-9aa is encoded by the nucleotide sequence of SEQ ID NO: 138 and said QsC28XylT4-12aa is encoded by the nucleotide sequence of SEQ ID NO: 141.

[0314] Clause 135. The method of clause 28 or clause 129, wherein the XylT is N-terminally tagged for solubility.

[0315] Clause 136. The method of clause 135, wherein said XylT is selected from SUMO-QsC28XylT4 as set forth in SEQ ID NO: 143, TrxA-QsC28-XylT4 as set forth in SEQ ID NO: 145, and MBP-QsC28XylT4 as set forth in SEQ ID NO: 147.

[0316] Clause 137. The method of clause 136, wherein said SUMO-QsC28XylT4 is encoded by the nucleotide sequence of SEQ ID NO: 144, said TrxA-QsC28-XylT4 is encoded by the nucleotide sequence of SEQ ID NO: 146, and said MBP-QsC28XylT4 is encoded by the nucleotide sequence of SEQ ID NO: 148.

[0317] Clause 138. The method of clause 128 or clause 129, wherein said XylT is QsC28XylT3-3xGGGS-QsC28XylT4 as set forth in SEQ ID NO: 149.

[0318] Clause 139. The method of clause 138, wherein said QsC28XylT3-3xGGGS-QsC28XylT4 is encoded by the nucleotide sequence of SEQ ID NO: 150.

[0319] Clause 140. A yeast recombinantly produced by the method according to any one of clauses 128 to 139, comprising QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl.

[0320] Clause 141. The C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api, and the overexpression is (iv) UDP-apiose synthase (AXS), which converts UDP-GlcA to UDP-Api, and / or (v) UDP-apiose transferase (ApiT), which transfers UDP-apiose and attaches the apiose residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api, respectively. 127. The method of any one of paragraphs 123 to 126, further comprising overexpressing a heterologous gene encoding the

[0321] Clause 142. The method of clause 141, wherein said AXS is QsAXS as set forth in SEQ ID NO:113.

[0322] Clause 143. The method of clause 142, wherein said QsAXS is encoded by the nucleotide sequence of SEQ ID NO:114.

[0323] Clause 144. The method according to any one of clauses 141 to 143, wherein said ApiT is selected from those derived from Q. saponaria (Qs) or S. vaccaria (Sv).

[0324] Clause 145. The method of clause 144, wherein said ApiT is QsC28ApiT4 as set forth in SEQ ID NO:151.

[0325] Clause 146. The method of clause 145, wherein said QsC28ApiT4 is encoded by the nucleotide sequence of SEQ ID NO:152.

[0326] Clause 147. A yeast recombinantly produced by the method according to any one of clauses 141 to 146, comprising a recombinant yeast capable of producing QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api.

[0327] 148. A method for producing (S)-2-methylbutyryl CoA (2MB-CoA) in yeast, comprising: In the presence of exogenously added 2-methylbutyric acid, (i) Carboxyl coenzyme A (CoA) ligase (CCL), which converts 2-methylbutyrate to 2MB-CoA The method comprises overexpressing a heterologous gene encoding the

[0328] Clause 149. The method of clause 148, wherein said CCL is QsCCL from Q. saponaria as set forth in SEQ ID NO:178.

[0329] Clause 150. The method of clause 149, wherein said QsCCL is encoded by the nucleotide sequence of SEQ ID NO:179.

[0330] Clause 151. The overexpression in the yeast, without exogenous addition of 2-methylbutyric acid, (ii) phosphopantetheinyl transferase (Ppant), and / or (iii) Megasynthetase LovF-TE containing an acyl carrier protein (ACP) domain and further comprising overexpressing a heterologous gene encoding 151. The method according to any one of items 148 to 150, wherein the megasynthetase LovF-TE condenses two units of malonyl-CoA with 2MB-ACP to cleave 2-methylbutyrate from the ACP domain and converts 2-methylbutyrate to 2MB-CoA by the CCL.

[0331] Clause 152. The method of clause 151, wherein said Ppant is from Aspergillus nidulans (An) and said megasynthase LovF-TE is from Aspergillus terreus (Ast).

[0332] Clause 153. The method of clause 152, wherein said Ppant is AnNpgA as set forth in SEQ ID NO: 237 and said megasynthase LovF-TE is AstLovF-TE as set forth in SEQ ID NO: 235.

[0333] Clause 154. The method of clause 153, wherein said AnNgA is encoded by the nucleotide sequence of SEQ ID NO: 238 and said AstLovF-TE is encoded by the nucleotide sequence of SEQ ID NO: 236.

[0334] Clause 155. A yeast recombinantly engineered to produce (S)-2-methylbutyryl-CoA (2MB-CoA) by the method of any one of clauses 148 to 154.

[0335] Item 156. A method for producing UDP-arabinofuranose (UDP-Araf) in yeast, comprising: In yeast engineered to produce UDP-Xyl, (i) a UDP-Xyl epimerase (UXE) that converts UDP-Xyl to UDP-arabinopyranose (UDP-Arap); and / or (ii) UDP-arabinose mutase (UAM), which converts UDP-Arap to UDP-arabinofuranose (UDP-Araf) The method comprises overexpressing a heterologous gene encoding the

[0336] Clause 157. The method of clause 156, wherein said UXE and said UAM are independently selected from those derived from A. thaliana (At) and H. vulgare (Hv).

[0337] Clause 158. The method of clause 157, wherein said UXE is selected from AtUXE as set forth in SEQ ID NO: 199, AtUXE2 as set forth in SEQ ID NO: 202, HvUXE-1 as set forth in SEQ ID NO: 240, HvUXE-2 as set forth in SEQ ID NO: 242, and AtUGE3 as set forth in SEQ ID NO: 205, and said UAM is selected from AtUAM1 as set forth in SEQ ID NO: 208, and HvUAM as set forth in SEQ ID NO: 211.

[0338] Clause 159. The method of clause 158, wherein said AtUXE is encoded by the nucleotide sequence of SEQ ID NO: 200, said AtUXE2 is encoded by the nucleotide sequence of SEQ ID NO: 203, said HvUXE-1 is encoded by the nucleotide sequence of SEQ ID NO: 241, said HvUXE-2 is encoded by the nucleotide sequence of SEQ ID NO: 243, said AtUAM1 is encoded by the nucleotide sequence of SEQ ID NO: 209, said HvUAM is encoded by the nucleotide sequence of SEQ ID NO: 212, and said AtUGE3 is encoded by the nucleotide sequence of SEQ ID NO: 206.

[0339] Clause 160. The method according to any one of clauses 156 to 159, wherein the yeast recombinantly produced UDP-Xyl is a yeast according to clause 62.

[0340] Paragraph 161. A yeast recombinantly produced to produce UDP-Araf by the method according to any one of paragraphs 156 to 160.

[0341] Item 162. A method for producing UDP-Araf in yeast, comprising: In the presence of exogenously added arabinose, (i) arabinokinase (AraK), and / or (ii) UDP-sugar pyrophosphorylase (USP) The method comprises overexpressing a heterologous gene encoding the

[0342] Clause 163. The method of clause 162, wherein said AraK and said USP are independently selected from those derived from A. thaliana (At) and pathogenic Leptospira interrogans (Lei).

[0343] Clause 164. The method of clause 163, wherein the AraK is selected from AtAraK as set forth in SEQ ID NO: 214 and LeiAraK as set forth in SEQ ID NO: 217, and the USP is selected from AtUSP as set forth in SEQ ID NO: 223 and LeiUSP as set forth in SEQ ID NO: 226.

[0344] Clause 165. The method of clause 164, wherein said AtAraK is encoded by the nucleotide sequence of SEQ ID NO: 215, said LeiAraK is encoded by the nucleotide sequence of SEQ ID NO: 218, said AtUSP is encoded by the nucleotide sequence of SEQ ID NO: 224, and said LeiUSP is encoded by the nucleotide sequence of SEQ ID NO: 227.

[0345] Clause 166. The method of any one of clauses 162 to 165, wherein said overexpressing further comprises (iii) overexpressing a heterologous gene encoding an arabinose transporter (AraT).

[0346] Clause 167. The method of clause 166, wherein the AraT is PrAraT from Penicillium rubens Wisconsin strain as set forth in SEQ ID NO:220.

[0347] Clause 168. The method of clause 167, wherein said PrAraT is encoded by the nucleotide sequence of SEQ ID NO:221.

[0348] Paragraph 169. A yeast recombinantly produced to produce UDP-Araf by the method according to any one of paragraphs 162 to 168.

[0349] Item 170. A method for producing an acylated glycosylated QA derivative in yeast, comprising: the acylated glycosylated QA derivative is QA-C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9, The method further comprising: In yeast engineered to produce QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXA or QA-C3-GGX-C28-FRXA, in the presence of exogenously added 2-methylbutyrate, (i) carboxyl coenzyme A ligase (CCL), which converts 2-methylbutyrate to (S)-2-methylbutyryl CoA (2MB-CoA); (ii) a chalcone synthase-like type III polyketide synthase (PKS) that condenses malonyl-CoA with 2MB-CoA to form C9-keto-CoA; (iii) a ketoreductase (KR) that converts C9-keto-CoA to C9-CoA; and / or (iv) an acyltransferase that transfers a first C9-CoA unit to combine with QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXA or QA-C3-GGX-C28-FRXA to form QA-C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9. overexpressing a heterologous gene encoding A method comprising:

[0350] Clause 171. The method of clause 170, wherein the CCL, the chalcone synthase-like type III PKS, the KR and the acyltransferase are derived from Q. saponaria.

[0351] Clause 172. The method of clause 171, wherein said CCL is QsCCL as set forth in SEQ ID NO: 178, said chalcone synthase-like type III PKS is QsChSD as set forth in SEQ ID NO: 181, QsChSE as set forth in SEQ ID NO: 184, or both QsChSD as set forth in SEQ ID NO: 181 and QsChSE as set forth in SEQ ID NO: 184, said ketoreductase is QsKR11 as set forth in SEQ ID NO: 187, QsKR23 as set forth in SEQ ID NO: 190, or both QsKR11 as set forth in SEQ ID NO: 187 and QsKR23 as set forth in SEQ ID NO: 190, and said acyltransferase is QsDMOT9 as set forth in SEQ ID NO: 193.

[0352] Clause 173. The method according to clause 171, wherein said chalcone synthase-like type III PKS is QsChSD as set forth in SEQ ID NO:181 and QsChSE as set forth in SEQ ID NO:184.

[0353] Clause 174. The method of clause 170, wherein said KR is QsKR11 as set forth in SEQ ID NO:187 and QsKR23 as set forth in SEQ ID NO:190.

[0354] Clause 175: The method according to any one of clauses 172 to 174, wherein the CCL is QsCCL as set forth in SEQ ID NO: 178, the chalcone synthase-like type III PKS is QsChSD as set forth in SEQ ID NO: 181 and QsChSE as set forth in SEQ ID NO: 184, the KR is QsKR11 as set forth in SEQ ID NO: 187 and QsKR23 as set forth in SEQ ID NO: 190, and the acyltransferase is QsDMOT9 as set forth in SEQ ID NO: 193.

[0355] Clause 176. The method of clause 175, wherein said QsCCL is encoded by SEQ ID NO: 179, said QsChSD is encoded by the nucleotide sequence of SEQ ID NO: 182, said QsChSE is encoded by the nucleotide sequence of SEQ ID NO: 185, said QsKR11 is encoded by the nucleotide sequence of SEQ ID NO: 188, said QsKR23 is encoded by the nucleotide sequence of SEQ ID NO: 191, and said QsDMOT9 is encoded by the nucleotide sequence of SEQ ID NO: 194.

[0356] Clause 177. The method according to any one of clauses 170 to 176, wherein the yeast recombinantly produced QA-C3-GGR-C28-FRX and QA-C3-GGX-C28-FRX is a yeast according to clause 127, the yeast recombinantly produced QA-C3-GGR-C28-FRXX and QA-C3-GGX-C28-FRXX is a yeast according to clause 140, and the yeast recombinantly produced QA-C3-GGR-C28-FRXA and QA-C3-GGX-C28-FRXA is a yeast according to clause 147.

[0357] Clause 178. Yeast engineered to produce QA-C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9 by the method according to any one of clauses 170 to 177.

[0358] Item 179. The acylated glycosylated QA derivative is QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXA-C18 or QA-C3-GGX-C28-FRXA-C18; The overexpression (v) attaching a second C9-CoA unit to C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9 to form C3-GGR- acyltransferase QsDMOT4 according to SEQ ID NO: 196 which forms C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXA-C18 or QA-C3-GGX-C28-FRXA-C18 179. The method of any one of paragraphs 170 to 178, further comprising overexpressing a heterologous gene encoding the

[0359] Clause 180. The method of clause 179, wherein said QsDMOT4 is encoded by the nucleotide sequence of SEQ ID NO:197.

[0360] Clause 181. Yeast engineered to produce QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRXA-C18 by the method according to clause 179 or clause 180.

[0361] Item 182. The acylated glycosylated QA derivative is QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf; the yeast is further engineered to produce UDP-Araf; The overexpression (vi) transferring UDP-Araf to attach Araf residues to QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXA-C18 or QA-C3-GGX-C28-FRXA-C18; arabinotransferase (ArafT) forming GR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf 179. The method of claim 180, further comprising overexpressing a heterologous gene encoding the

[0362] Clause 183. The method of clause 182, wherein said ArafT is derived from Q. saponaria (Qs).

[0363] Clause 184. The method of clause 182 or clause 183, wherein said ArafT is selected from QsArafT as set forth in SEQ ID NO:229, and QsArafT2 as set forth in SEQ ID NO:232.

[0364] Clause 185. The method of clause 184, wherein said QsArafT is encoded by the nucleotide sequence of SEQ ID NO:230 and said QsArafT2 is encoded by the nucleotide sequence of SEQ ID NO:233.

[0365] Clause 186. The method of clause 184, wherein said ArafT is QsArafT2 as set forth in SEQ ID NO:232.

[0366] Clause 187. The method of clause 186, wherein said QsArafT2 is encoded by the nucleotide sequence of SEQ ID NO:233.

[0367] Clause 188. The method according to any one of clauses 182 to 187, wherein said yeast engineered to produce UDP-Araf is a yeast according to clause 161 or clause 169.

[0368] Clause 189. A yeast engineered to produce QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf by the method according to any one of clauses 182 to 188.

[0369] Item 190. A method for producing QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GGR-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXA-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl in yeast, comprising: in a yeast engineered to produce QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GRX-C28-FRXX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRX-C18, By transferring UDP-Xyl to QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GRX-C28-FRXX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRX-C18, a Xyl residue was attached to QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, or QA-C3-GGR-C28-FRX-C18. overexpressing a heterologous gene encoding an arabinotransferase (ArafT) that forms 8-Xyl, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GRX-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXA-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl A method comprising:

[0370] Clause 191. The method of clause 190, wherein said ArafT is QsArafT as set forth in SEQ ID NO:229.

[0371] Clause 192. The method of clause 191, wherein said QsArafT is encoded by the nucleotide sequence of SEQ ID NO:230.

[0372] Clause 193. The method according to any one of clauses 190 to 192, wherein the yeast recombinantly produced to produce QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GRX-C28-FRXX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRX-C18 is a yeast according to clause 181.

[0373] Clause 194. A yeast engineered to produce QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GRX-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXA-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl by the method according to any one of clauses 190 to 193.

[0374] Clause 195: The overexpression is carried out without exogenous addition of 2-methylbutyric acid, (vii) phosphopantetheinyl transferase (Ppant), and / or (viii) Megasynthetase LovF-TE containing an acyl carrier protein (ACP) domain and further comprising overexpressing a heterologous gene encoding The method according to any one of paragraphs 170 to 177, 179 to 180, 182 to 188 and 190 to 193, wherein the megasynthetase LovF-TE condenses two units of malonyl-CoA with 2MB-ACP, cleaves 2-methylbutyrate from the ACP domain and converts 2-methylbutyrate to 2MB-CoA by CoA ligase (CCL).

[0375] Clause 196. The method of clause 195, wherein said Ppant is from Aspergillus nidulans (An) and said megasynthase LovF-TE is from Aspergillus terreus (Ast).

[0376] Clause 197. The method of clause 196, wherein said Ppant is AnNpgA as set forth in SEQ ID NO: 237 and said megasynthase LovF-TE is AstLovF-TE as set forth in SEQ ID NO: 235.

[0377] Clause 198. The method of clause 197, wherein said AnNgA is encoded by the nucleotide sequence of SEQ ID NO: 238 and said AstLovF-TE is encoded by the nucleotide sequence of SEQ ID NO: 236.

[0378] Clause 199. A yeast engineered to produce QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf by the method according to any one of clauses 195 to 198.

[0379] 200. A method for producing QA-C3-GGX-C28-FRXX-C18-Araf (QS-21-Xyl) in yeast, comprising: GvBAS shown in SEQ ID NO: 10, QsC28C16 shown in SEQ ID NO: 23, QsC23 shown in SEQ ID NO: 29, QsC28 shown in SEQ ID NO: 41, AtATR1 shown in SEQ ID NO: 49, Qsb5 shown in SEQ ID NO: 55, SvMSBP1 shown in SEQ ID NO: 67, AtUGD shown in SEQ ID NO: 108 A101L, QsCslG2 as shown in SEQ ID NO: 78, QsGalT as shown in SEQ ID NO: 116, AtUXS as shown in SEQ ID NO: 105, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFuct as shown in SEQ ID NO: 93, AtRHM2 as shown in SEQ ID NO: 102, QsRhaT as shown in SEQ ID NO: 119, QsC28XylT3 as shown in SEQ ID NO: 125, QsC28XylT4 as shown in SEQ ID NO: 128, QsChSD as shown in SEQ ID NO: 181, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFuct as shown in SEQ ID NO: 93, AtRHM2 as shown in SEQ ID NO: 102, QsRhaT as shown in SEQ ID NO: 119, QsC28XylT3 as shown in SEQ ID NO: 125, QsC28XylT4 as shown in SEQ ID NO: 128, QsChSD as shown in SEQ ID NO: 18 4, QsChSE as set forth in SEQ ID NO: 187, QsKR11 as set forth in SEQ ID NO: 190, QsDMOT9 as set forth in SEQ ID NO: 193, QsDMOT4 as set forth in SEQ ID NO: 196, AtUXE as set forth in SEQ ID NO: 199, AtUAM1 as set forth in SEQ ID NO: 208, QsArafT2 as set forth in SEQ ID NO: 232, AnNpgA as set forth in SEQ ID NO: 237, QsCCL as set forth in SEQ ID NO: 178 and / or AstLovF-TE as set forth in SEQ ID NO: 235.

[0380] Paragraph 201, the GvBAS is encoded by a nucleotide sequence of SEQ ID NO: 11, the QsC28C16 is encoded by a nucleotide sequence of SEQ ID NO: 24, the QsC23 is encoded by a nucleotide sequence of SEQ ID NO: 30, the QsC28 is encoded by a nucleotide sequence of SEQ ID NO: 42, the AtATR1 is encoded by a nucleotide sequence of SEQ ID NO: 50, the Qsb5 is encoded by a nucleotide sequence of SEQ ID NO: 56, the SvMSBP1 is encoded by a nucleotide sequence of SEQ ID NO: 68, and the AtUGD A101Lis encoded by the nucleotide sequence of SEQ ID NO: 109, QsCslG2 is encoded by the nucleotide sequence of SEQ ID NO: 82, QsGalT is encoded by the nucleotide sequence of SEQ ID NO: 117, AtUXS is encoded by the nucleotide sequence of SEQ ID NO: 106, QsC3XylT is encoded by the nucleotide sequence of SEQ ID NO: 123, SvNMD is encoded by the nucleotide sequence of SEQ ID NO: 91, SvUG46DH is encoded by the nucleotide sequence of SEQ ID NO: 88, QsFucT is encoded by the nucleotide sequence of SEQ ID NO: 94, AtRHM2 is encoded by the nucleotide sequence of SEQ ID NO: 103, QsRhaT is encoded by the nucleotide sequence of SEQ ID NO: 220, QsC28XylT3 is encoded by the nucleotide sequence of SEQ ID NO: 126, QsC28XylT4 is encoded by the nucleotide sequence of SEQ ID NO: 129, and QsChSD is The method of claim 200, wherein the QsChSE is encoded by the nucleotide sequence of SEQ ID NO: 182, the QsKR11 is encoded by the nucleotide sequence of SEQ ID NO: 188, the QsKR23 is encoded by the nucleotide sequence of SEQ ID NO: 191, the QsDMOT9 is encoded by the nucleotide sequence of SEQ ID NO: 194, the QsDMOT4 is encoded by the nucleotide sequence of SEQ ID NO: 197, the AtUXE is encoded by the nucleotide sequence of SEQ ID NO: 200, the AtUAM1 is encoded by the nucleotide sequence of SEQ ID NO: 209, the QsArafT2 is encoded by the nucleotide sequence of SEQ ID NO: 233, the AnNpgA is encoded by the nucleotide sequence of SEQ ID NO: 238, the QsCCL is encoded by the nucleotide sequence of SEQ ID NO: 179, and the AstLovF-TE is encoded by the nucleotide sequence of SEQ ID NO: 236.

[0381] Clause 202. A yeast recombinantly engineered to produce QS-21-Xyl by the method of clause 201 or clause 202.

[0382] 203. A method for producing QA-C3-GGX-C28-FRXA-C18-Araf (QS-21-Api) in yeast, comprising: GvBAS shown in SEQ ID NO: 10, QsC28C16 shown in SEQ ID NO: 23, QsC23 shown in SEQ ID NO: 29, QsC28 shown in SEQ ID NO: 41, AtATR1 shown in SEQ ID NO: 49, Qsb5 shown in SEQ ID NO: 55, SvMSBP1 shown in SEQ ID NO: 67, AtUGD shown in SEQ ID NO: 108 A101L , QsCslG2 as shown in SEQ ID NO: 81, QsGalT as shown in SEQ ID NO: 116, AtUXS as shown in SEQ ID NO: 105, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFucT as shown in SEQ ID NO: 93, AtRHM2 as shown in SEQ ID NO: 102, QsRhaT as shown in SEQ ID NO: 119, QsC28XylT3 as shown in SEQ ID NO: 125, QsC28ApiT4 as shown in SEQ ID NO: 151, QsChSD as shown in SEQ ID NO: 181, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFucT as shown in SEQ ID NO: 93, 4, QsChSE as set forth in SEQ ID NO: 187, QsKR11 as set forth in SEQ ID NO: 190, QsDMOT9 as set forth in SEQ ID NO: 193, QsDMOT4 as set forth in SEQ ID NO: 196, AtUXE as set forth in SEQ ID NO: 199, AtUAM1 as set forth in SEQ ID NO: 208, QsArafT2 as set forth in SEQ ID NO: 232, AnNpgA as set forth in SEQ ID NO: 237, QsCCL as set forth in SEQ ID NO: 178 and / or AstLovF-TE as set forth in SEQ ID NO: 235.

[0383] Clause 204: The GvBAS is encoded by a nucleotide sequence of SEQ ID NO: 11, the QsC28C16 is encoded by a nucleotide sequence of SEQ ID NO: 24, the QsC23 is encoded by a nucleotide sequence of SEQ ID NO: 30, the QsC28 is encoded by a nucleotide sequence of SEQ ID NO: 42, the AtATR1 is encoded by a nucleotide sequence of SEQ ID NO: 50, the Qsb5 is encoded by a nucleotide sequence of SEQ ID NO: 56, the SvMSBP1 is encoded by a nucleotide sequence of SEQ ID NO: 68, and the AtUGDA101L is encoded by the nucleotide sequence of SEQ ID NO: 109, QsCslG2 is encoded by the nucleotide sequence of SEQ ID NO: 82, QsGalT is encoded by the nucleotide sequence of SEQ ID NO: 117, AtUXS is encoded by the nucleotide sequence of SEQ ID NO: 106, QsC3XylT is encoded by the nucleotide sequence of SEQ ID NO: 123, SvNMD is encoded by the nucleotide sequence of SEQ ID NO: 91, SvUG46DH is encoded by the nucleotide sequence of SEQ ID NO: 88, QsFucT is encoded by the nucleotide sequence of SEQ ID NO: 94, AtRHM2 is encoded by the nucleotide sequence of SEQ ID NO: 103, QsRhaT is encoded by the nucleotide sequence of SEQ ID NO: 120, QsC28XylT3 is encoded by the nucleotide sequence of SEQ ID NO: 126, QsC28ApiT4 is encoded by the nucleotide sequence of SEQ ID NO: 152, and QsChSD is The method of claim 203, wherein the QsChSE is encoded by the nucleotide sequence of SEQ ID NO: 182, the QsKR11 is encoded by the nucleotide sequence of SEQ ID NO: 188, the QsKR23 is encoded by the nucleotide sequence of SEQ ID NO: 191, the QsDMOT9 is encoded by the nucleotide sequence of SEQ ID NO: 194, the QsDMOT4 is encoded by the nucleotide sequence of SEQ ID NO: 197, the AtUXE is encoded by the nucleotide sequence of SEQ ID NO: 200, the AtUAM1 is encoded by the nucleotide sequence of SEQ ID NO: 209, the QsArafT2 is encoded by the nucleotide sequence of SEQ ID NO: 233, the AnNpgA is encoded by the nucleotide sequence of SEQ ID NO: 238, the QsCCL is encoded by the nucleotide sequence of SEQ ID NO: 179, and the AstLovF-TE is encoded by the nucleotide sequence of SEQ ID NO: 236.

[0384] Clause 205. A yeast recombinantly produced by the method of clause 204 or clause 205.

[0385] Clause 206. The GvBAS (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 10, the QsC28C16 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 23, the QsC23 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 29, and the QsC28 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 41. %, 95%, 98% or 99% identity to SEQ ID NO: 49, said AtATR1 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 49, said Qsb5 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 55, said SvMSBP1 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 67, and said AtUGD A101L(if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 108, said QsCslG2 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 81, said QsGalT (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 116, and said AtUXS (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 105. %, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 122, said QsC3XylT (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 122, said SvNMD (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 90, and said SvUG46DH (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 87. The QsFucT (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 93, the AtRHM2 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 102, the QsC28XylT3 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 125, and the Qs C28XylT4 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 128, said QsC28ApiT4 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 151, said QsChSD (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 181, and said QsChSE is represented by SEQ ID NO: 184;The QsKR11 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 187, the QsKR23 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 190, the QsDMOT9 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 193, and the QsDMOT4 (if present) is represented by the sequence The AtUXE (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 199, the AtUAM1 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 208, and the QsArafT2 (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 232. , 95%, 98% or 99% identity to SEQ ID NO: 237, wherein the AnNpgA (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 237, wherein the QsCCL (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 178, and wherein the AstLovF-TE (if present) is represented by an amino acid sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 235. 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188,A method according to any one of paragraphs 190 to 193, 195 to 198, 200, 201, 203 and 204, or a yeast according to any one of paragraphs 39, 40, 46, 51, 62, 73, 81, 87, 92, 101, 109, 116, 117, 122, 127, 140, 147, 155, 161, 169, 178, 181, 189, 194, 199, 202 and 205.

[0386] Clause 207: Clauses 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182, wherein the heterologous gene is integrated into the genome of the yeast. A method according to any one of paragraphs 39, 40, 46, 51, 62, 73, 81, 87, 92, 101, 109, 116, 117, 122, 127, 140, 147, 155, 161, 169, 178, 181, 189, 194, 199, 202, 205 and 206.

[0387] Clause 208. The method or yeast according to clause 207, wherein any one or more of said heterologous genes is integrated in one or more copies.

[0388] Item 209. The method or yeast according to item 208, wherein said one or more copies is between two and five copies.

[0389] Clause 210. The method or yeast of clause 208 or clause 209, wherein at least two copies of the genes encoding the C16 oxidase, the C23 oxidase and / or the C28 oxidase are integrated.

[0390] Clause 211. The method or yeast according to any one of clauses 208 to 210, wherein the gene encoding said UXS (if present) is integrated in at least three copies.

[0391] Clause 212. The method or yeast according to clause 208 to 211, wherein the nucleotide sequence of the heterologous gene is codon-optimized.

[0392] Clause 213. The GvBAS (if present) is encoded by a nucleotide sequence of SEQ ID NO: 12, the QsC28C16 (if present) is encoded by a nucleotide sequence of SEQ ID NO: 25, the QsC23 (if present) is encoded by a nucleotide sequence of SEQ ID NO: 31, the QsC28 (if present) is encoded by a nucleotide sequence of SEQ ID NO: 43, the AtATR1 (if present) is encoded by a nucleotide sequence of SEQ ID NO: 51, the Qsb5 (if present) is encoded by a nucleotide sequence of SEQ ID NO: 57, the SvMSBP1 is encoded by a nucleotide sequence of SEQ ID NO: 69, and the AtUGD A101L(if present) is encoded by the nucleotide sequence of SEQ ID NO: 109, said QsCslG2 (if present) is encoded by the nucleotide sequence of SEQ ID NO: 83, said QsGalT (if present) is encoded by the nucleotide sequence of SEQ ID NO: 118, said AtUXS (if present) is encoded by the nucleotide sequence of SEQ ID NO: 107, said QsC3XylT (if present) is encoded by the nucleotide sequence of SEQ ID NO: 124, and said SvNMD (if present) is encoded by the nucleotide sequence of SEQ ID NO: 92. wherein said SvUG46DH (if present) is encoded by the nucleotide sequence of SEQ ID NO:89, said QsFucT (if present) is encoded by the nucleotide sequence of SEQ ID NO:95, said AtRHM2 (if present) is encoded by the nucleotide sequence of SEQ ID NO:104, said QsRhaT (if present) is encoded by the nucleotide sequence of SEQ ID NO:121, said QsC28XylT3 (if present) is encoded by the nucleotide sequence of SEQ ID NO:127, and said QsC28XylT4 (if present) is encoded by the nucleotide sequence of SEQ ID NO: QsC28ApiT4 (if present) is encoded by the nucleotide sequence of SEQ ID NO: 153, QsChSD (if present) is encoded by the nucleotide sequence of SEQ ID NO: 183, QsChSE (if present) is encoded by the nucleotide sequence of SEQ ID NO: 186, QsKR11 (if present) is encoded by the nucleotide sequence of SEQ ID NO: 189, QsKR23 (if present) is encoded by the nucleotide sequence of SEQ ID NO: 192, and QsDM OT9 (if present) is encoded by the nucleotide sequence of SEQ ID NO: 195, QsDMOT4 is encoded by the nucleotide sequence of SEQ ID NO: 198, AtUXE (if present) is encoded by the nucleotide sequence of SEQ ID NO: 201, AtUAM1 (if present) is encoded by the nucleotide sequence of SEQ ID NO: 210, QsArafT2 (if present) is encoded by the nucleotide sequence of SEQ ID NO: 234, and AnNpgA (if present) is encoded by the nucleotide sequence of SEQ ID NO: 239;213. The method or yeast according to paragraph 212, wherein said QsCCL, if present, is encoded by the nucleotide sequence of SEQ ID NO: 180, and said AstLovF-TE, if present, is encoded by the nucleotide sequence of SEQ ID NO: 236.

[0393] Item 214: The method according to items 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 12 to 13 further comprises a step of culturing the yeast to produce queratic acid (QA), each UDP-sugar and / or each QA derivative. The method according to any one of paragraphs 3 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188, 190 to 193, 195 to 198, 200, 201, 203, 204, and 206 to 213.

[0394] Item 215. The method according to item 214, wherein the culturing step is carried out for 2 to 6 days.

[0395] Clause 216. The method of clause 215, wherein said culturing step is carried out for about 3 days.

[0396] Section 217 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 184, 188, 190 to 193, 195 to 198, 200, 201, 203, 204 and 206 to 216, or the yeast according to any one of 39, 40, 46, 51, 62, 73, 81, 87, 92, 101, 109, 116, 117, 122, 127, 140, 147, 155, 161, 169, 178, 181, 189, 194, 199, 202 and 205 to 213.

[0397] Clause 218. The method or yeast according to clause 217, wherein induction of the promoter is carried out for 2 to 5 days and the yeast is cultured for an additional 2 to 5 days.

[0398] Item 219: The method according to any one of items 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, or 116, further comprising a step of isolating the UDP-sugar, the C3-glycosylated QA derivative, the C28-glycosylated QA derivative, or the acylated glycosylated QA derivative. The method according to any one of paragraphs 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188, 190 to 193, 195 to 198, 200, 201, 203, 204, and 206 to 218.

[0399] Item 220. Queric acid (QA) obtained by the method according to any one of items 1 to 38.

[0400] Item 221. A C3-glycosylated queratic acid (QA) derivative, a C28-glycosylated QA derivative, or an acylated glycosylated QA derivative, obtainable by the method according to item 219.

[0401] Item 222. Use of a quillaric acid (QA) derivative according to item 221 as an adjuvant.

[0402] Clause 223. The use of clause 222, wherein the adjuvant is a liposomal formulation.

[0403] Clause 224. The use of clause 222 or clause 223, wherein the adjuvant comprises a TLR4 agonist.

[0404] Clause 225. The use of clause 224, wherein said TLR4 agonist is 3D-MPL.

[0405] Clause 226: An adjuvant composition comprising QS-21-Xyl according to clause 201 and / or QS-21-Api according to clause 203.

[0406] Item 227. An isolated β-amyrin synthase (SvBAS) as set forth in SEQ ID NO:13.

[0407] Item 228. An isolated β-amyrin synthase (QsBAS) as set forth in SEQ ID NO:15.

[0408] Item 229. An isolated CYP C16 oxidase (QsC28C16) as set forth in SEQ ID NO:23.

[0409] Item 230. An isolated CYP C16 oxidase (SvC16) as set forth in SEQ ID NO:26.

[0410] Item 231. An isolated CYP C23 oxidase (SvC23-1) as set forth in SEQ ID NO:32.

[0411] Item 232. An isolated CYP C23 oxidase (SvC23-2) as set forth in SEQ ID NO:35.

[0412] Item 233. An isolated CYP C28 oxidase (SvC28) as set forth in SEQ ID NO:44.

[0413] Item 234. An isolated cytochrome b5 protein (Qsb5) as set forth in SEQ ID NO:55.

[0414] Item 235. An isolated cytochrome b5 protein (Svb5) as set forth in SEQ ID NO:61.

[0415] Item 236. An isolated UDP-GlcA transferase (SvCslG) as set forth in SEQ ID NO: 76.

[0416] Item 237. The isolated MSBP protein (SvMSBP1) as set forth in SEQ ID NO:67.

[0417] Item 238. The isolated MSBP protein (SvMSBP2) as set forth in SEQ ID NO: 70.

[0418] Item 239. The isolated MSBP protein (QsMSBP1) as set forth in SEQ ID NO:73.

[0419] Item 240. An isolated UDP-glucose-4,6-dehydratase (SvUG46DH) as set forth in SEQ ID NO:87.

[0420] Item 241. An isolated UDP-4-keto-6-deoxyglucose reductase (SvNMD) as set forth in SEQ ID NO:90.

[0421] Item 242. An isolated UDP-galactose transferase (SvGalT) as set forth in SEQ ID NO:98.

[0422] Item 243. An isolated UDP-fucosyltransferase (SvFucT) as set forth in SEQ ID NO:96.

[0423] Item 244. An isolated UDP-xylose transferase (SvC3XylT) as set forth in SEQ ID NO:100.

[0424] Item 245. An isolated UDP-arabinofuranose transferase (QsArafT2) as set forth in SEQ ID NO: 229.

[0425] 246. The isolated UDP-glucose dehydrogenase (AtUGD) as set forth in SEQ ID NO: 108 A101L ).

[0426] Item 247. An isolated UDP-xylosetransferase (QsC28XylT4-3aa) as set forth in SEQ ID NO: 131.

[0427] Item 248. An isolated UDP-xylosetransferase (QsC28XylT4-6aa) as set forth in SEQ ID NO: 134.

[0428] Item 249. An isolated UDP-xylosetransferase (QsC28XylT4-9aa) as set forth in SEQ ID NO: 137.

[0429] Item 250. An isolated UDP-xylosetransferase (QsC28XylT4-12aa) as set forth in SEQ ID NO: 140.

[0430] Item 251. An isolated UDP-xylosyltransferase (SUMO-QsC28XylT4) as represented by SEQ ID NO: 143.

[0431] Item 252. An isolated UDP-xylose transferase (TrXA-QsC28XylT4) as set forth in SEQ ID NO: 145.

[0432] Item 253. An isolated UDP-xylosetransferase (MBP-QsC28XylT4) as set forth in SEQ ID NO: 147.

[0433] Item 254. The isolated UDP-xylose transferase as set forth in SEQ ID NO: 149 (QsC28XylT3-3xGGGS-QsC28XylT4).

[0434] Item 255. An isolated type I polyketide synthase (AstLovF-TE) as set forth in SEQ ID NO: 235. EXAMPLES

[0435] The genotypes of the YL yeast strains used in the examples described below are shown in Table 3, and the genotypes of the SC yeast strains are shown in Table 4. Genetic modification of yeast was performed as described in Example 5 below (unless otherwise stated). To enhance the production of the protein corresponding to each heterologous gene, a codon-optimized nucleotide sequence was used to express each heterologous gene in yeast. Codon optimization does not affect the amino acid sequence of the overexpressed protein. Unless otherwise stated, the integration of the various heterologous genes into the genome of the various yeast strains was performed in the presence of a galactose-inducible promoter (as shown in the examples below). After 2 days of cultivation, the expression of the heterologous genes was induced by adding galactose to the culture medium. Unless otherwise stated, 3 days after induction of expression, production of sugars, quillic acid precursors, quillic acid (QA), and acylated and / or glycosylated QA derivatives (described below) was assessed by extraction from yeast culture medium (as described in Example 5 below) and analysis for their presence by liquid chromatography-mass spectrometry (LC-MS) (as described in Example 6 below).

[0436] Example 1 – Biosynthesis of Quillaric Acid (QA) 1.1 Generation of β-amyrin precursor The previously developed mevalonate-overproducing Jwy601 strain (a Saccharomyces cerevisiae strain derived from the CEN.PK2 yeast strain) was selected as the parent strain (Wong et al., 2018). Various recombinant yeast strains were generated by engineering Jwy601 strain to overexpress genes encoding β-amyrin synthases (BAS) from various types of plants through genomic integration, and these yeast strains were tested for their ability to convert 2,3-oxidosqualene to β-amyrin by analyzing the presence or absence of β-amyrin by gas chromatography-mass spectrometry (GC-MS) (using commercially available standards).

[0437] result In this study, β-amyrin synthase (BAS) from Artemisia annua (Aa) (referred to as the "AaBAS" enzyme - SEQ ID NO: 1 encoded by SEQ ID NO: 3), β-amyrin synthase (BAS) from Arabidopsis thaliana (At) (referred to as the "AtBAS" enzyme - SEQ ID NO: 4 encoded by SEQ ID NO: 6), β-amyrin synthase (BAS) from Glycyrrhiza glabra (Gg) (referred to as the "GgBAS" enzyme - SEQ ID NO: 7 encoded by SEQ ID NO: 9), and β-amyrin synthase (BAS) from Gypsophila vaccaria (Gv) (referred to as the "GvBAS" enzyme - SEQ ID NO: 10 encoded by SEQ ID NO: 12) were tested. The BAS homologue from Gypsophila produced the highest amount of β-amyrin (see FIG. 3). With this in mind, a yeast strain engineered to express GvBAS (MLY-01) was selected for further engineering as described below.

[0438] 1.2 QA Generation and Generation Optimization 1.2.1 Production of QA precursors in yeast MLY-01 was further engineered to co-express various cytochrome P450 (CYP) oxidases (C16 oxidase, C23 oxidase, or C28 oxidase) from different plants and cytochrome P450 reductase (CPR) by sequential integration into the yeast genome. The production of QA and QA precursors was analyzed by LC-MS (using the respective commercial standards, e.g., Merck standards, as reference) in yeast strains carrying the following enzyme combinations: CPR from Arabidopsis thaliana (referred to as "AtATR1" - SEQ ID NO: 49, encoded by SEQ ID NO: 51) C16 oxidase from Bupleurum falcatum [CYP716Y1] (referred to as "BfC16" - SEQ ID NO: 17 encoded by SEQ ID NO: 19) C23 oxidase from Medicago truncatula [CYP72A68] (referred to as "MtC23" - SEQ ID NO: 38, encoded by SEQ ID NO: 40) C28 oxidase from Medicago sativa [CYP716A12] (referred to as "MtC28" - SEQ ID NO: 46 encoded by SEQ ID NO: 48)

[0439] result Hederagenin and gypsogenin (QA precursors) were detectable, and images taken at approximately 10 min showed traces (<1 mg / L) of QA (data not shown; data are disclosed in Figure 3 of WO20 / 26354). These results demonstrate the functional association and activity of CPR and CYP oxidase expressed in yeast, and confirm the ability to generate QA when co-expressed in yeast.

[0440] To further test CYP oxidases from other plants, MLY-01 was further engineered to co-express a CYP oxidase homologue from Quillaja japonica and the AtAtr1 gene by sequential integration into the yeast genome. C16 oxidase [CYP716A297] (referred to as "QsC16" - SEQ ID NO:20 encoded by SEQ ID NO:22) C23 oxidase [CYP714E52] (referred to as "QsC23" - SEQ ID NO:29 encoded by SEQ ID NO:31) C28 oxidase [CYP716A24] (referred to as "QsC28" - SEQ ID NO: 41 encoded by SEQ ID NO: 43)

[0441] In some experiments, the cytochrome b5 protein from Quillaja japonica (referred to as "Qsb5" - SEQ ID NO: 55, encoded by SEQ ID NO: 57) and / or the steroid-binding membrane protein from Saponaria vacccaria (referred to as "SvMSBP1" - SEQ ID NO: 67, encoded by SEQ ID NO: 69) were additionally co-expressed (see also Sections 1.2.4 and 1.2.5 below).

[0442] The production of QA and QA precursors in yeast strains incorporating the following enzyme combinations was analyzed by LC-MS (using the respective commercial standards, e.g. from Merck, as reference): AtATR1-QsC28(YL-1) ·AtATR1-QsC28-QsC23-Qsb5(YL-3) ·AtATR1-QsC28-QsC23-Qsb5-QsC28C16(YL-4) ·AtATR1-QsC28-QsC23-Qsb5-QsC28C16-SvMSBP1(YL-6) AtATR1 (2 copies) - QsC28 (2 copies) - QsC23 - Qsb5 - QsC28C16 (YL-8) AtATR1 (2 copies) - QsC28 (2 copies) - QsC23 (2 copies) - Qsb5 (2 copies) - QsC28C16 (2 copies) - SvMSBP1 (2 copies) (YL-10)

[0443] The data is shown below in Table 2 and the results are depicted graphically in FIG.

[0444] [Table 2]

[0445] result As shown in Figure 4 and Table 2, AtATR1 (CPR reductase) alone was sufficient to oxidize C28 to form a carboxylic acid, producing 263.4 mg / L of oleanolic acid (YL-1), whereas oxidation of C23 by cytochrome b5 (Qsb5) from Quillaja japonica was required to oxidize the hydroxyl group at C23 to convert it to the aldehyde functional group of gypsogenin (YL-3).

[0446] Further coexpression of QsC16 (with AtATR1, QsC23, and QsC28) did not produce QA (data not shown), indicating that oxidation did not occur at C16, suggesting that QsC16 is not functional.

[0447] Investigating the intracellular localization of QsC16 oxidase tagged with mCherry at its C-terminus revealed that, unlike other CYP oxidases, QsC16 oxidase is localized in the cytosol, although it is predicted to have a transmembrane domain at its N-terminus. The confocal microscopy images obtained show that QsC18-GFP is localized to the membrane of the endoplasmic reticulum (ER) (Figure 5, left image), whereas QsC16-mCherry is localized in the cytosol (Figure 5, center image).

[0448] To test the hypothesis that the lack of activity of QsC16 was due to inappropriate localization in yeast, we re-examined the N-terminus of QsC16 by fusing the predicted 22 amino acid transmembrane domain of QsC28 to the N-terminus of QsC16 (this fusion is called "QsC28C16" - sequence number 23 encoded by sequence number 25). This transmembrane domain was shown to tether QsC16 to the endoplasmic reticulum membrane where CPR, other CYP oxidases, and the terpene substrate (β-amyrin) co-localize (Figure 5, right image) (data not shown).

[0449] Furthermore, when QsC28C16 (instead of QsC16) was coexpressed in YL-4, QA was detected at a production level of 1.1 mg / L (see Table 2 and FIG. 4).

[0450] Furthermore, co-expression of SvMSBP1 in YL-6 increased the overall oxidation efficiency and enhanced the production of QA (see Table 2 and Figure 4). Although the total titer of QA precursors remained constant, co-expression of SvMSBP1 increased the production of the final oxidation product (QA) four-fold (4 mg / L), and SvMSBP1 was co-localized in the endoplasmic reticulum membrane with QsC28 oxidase and QsC23 oxidase (data not shown).

[0451] Moreover, when two copies of QsC28 and two copies of AtATR1 were simultaneously overexpressed in YL-8, the amount of QA produced increased eight-fold (18.9 mg / L) (see Table 2 and Figure 4).

[0452] Moreover, the addition of two copies of all enzymes in YL-10 further optimized the QA production (65.2 mg / L) (see Table 2 and Figure 4).

[0453] 1.2.2 Genetic search in Saponaria vacccaria - CYP oxidase Saponaria baccaria (Sv) leaves and flowers were treated with 0.50 μM or 100 μM methyl jasmonate (MeJa) for 72 h. The expression levels of β-amyrin synthase mRNA (in leaves) were analyzed (see Figure 6A) and the fold change of β-amyrin synthase mRNA expression in flowers induced with 50 μM or 100 μM MeJa was examined at 24 and 72 h compared to 0 μM (see Figure 6B). Figure 6C shows a neighbor-joining phylogenetic tree (bootstrap analysis with 1000 replicates) of cytochrome P450 (CYP) oxidases from other plants acting on triterpenoids and CYP candidates identified from the transcriptome of Saponaria baccaria (see also Section 1.2.4 below). The gene names of the newly identified CYPs from Saponaria baccaria are marked with an asterisk (*). The gene names of the newly identified CYPs from Saponaria baccaria that are co-expressed with β-amyrin synthase (BAS) are boxed.

[0454] "SvC16", "SvC23-1", "Sv23-2" and "SvC28" (designated in Figure 6C) were combined with β-amyrin synthases (BAS) from various plants to test their functional relevance and activity in N. benthamiana. Various combinations of the following enzymes were transiently expressed in N. benthamiana (shown in Figure 7): β-amyrin synthase (BAS) from Saponaria vacccaria (referred to as "SvBAS" - SEQ ID NO:13 encoded by SEQ ID NO:14) β-amyrin synthase (BAS) from Quillaja japonica (referred to as "QsBAS" - SEQ ID NO:15 encoded by SEQ ID NO:16) C16 oxidase from Saponaria vaccaria (referred to as "SvC16" - SEQ ID NO:26 encoded by SEQ ID NO:27) C28 oxidase from Saponaria vacccaria (referred to as "SvC28" - SEQ ID NO: 44 encoded by SEQ ID NO: 45) C23 oxidase from Saponaria vaccaria (designated "SvC23-1" - SEQ ID NO:32 encoded by SEQ ID NO:33) C23 oxidase from Saponaria vaccaria (designated "SvC23-2" - SEQ ID NO:35 encoded by SEQ ID NO:36)

[0455] The production of QA precursors was analyzed by LC-MS (using commercially available standards, eg, standards available from MCE, Chemcruz or TCI, as references).

[0456] result The results are shown in Figure 7. · When SvBAS, SvC28, and SvC16 were coexpressed, echinocystic acid and oleanolic acid were detected (Fig. 7A). · When QsBAS and QsC28 were coexpressed with SvC23-1 or SvC23-2, gypsogenin was detected (Fig. 7B). · When QsBAS and QsC28 were coexpressed with SvC23-1 or SvC23-2, gypsogenic acid was detected (Fig. 7C). These results demonstrate the functional association and activities of QsBAS and the newly identified SvC16 oxidase, SvC23-1 oxidase, SvC23-2 oxidase and / or SvC28 oxidase, and confirm their ability to generate QA precursors when coexpressed in N. benthamiana.

[0457] 1.2.3 Generation of QA in yeast using the SvC16, SvC23-1 and SvC23-2 genes of Saponaria vacccaria MLY-01 was transformed with the following plasmids: pESC-TRP-SepGAL2-SvC16, pGAL10-AtAtr1, pGAL1-QsC28, and pGAL7-SvC23-1, or pESC-TRP-SepGAL2-SvC16, pGA10-AtAtr1, pGAL1-QsC28, and pGAL7-SvC23-2. The production of QA and QA precursors was analyzed by HPLC / LC-MS (using commercial standards as references).

[0458] result The chromatograms in Figures 8 and 9 show peaks that perfectly match the precise m / z values ​​and retention times of the commercially available QA standards (dashed lines). Confocal microscopy images revealed that SvC16, unlike QsC16 (see Section 1.2.1 above), was well expressed and properly localized to the endoplasmic reticulum in yeast (data not shown). These results indicate the functional relevance of SvC16 oxidase, SvC23-1 oxidase and / or SvC23-2 oxidase and confirm that they can produce QA when coexpressed with AtATR1 and QsC28 in yeast.

[0459] 1.2.4 Genetic search in Saponaria vacccaria - MSBP protein Genes encoding homologs of MSBPs from Arabidopsis thaliana (At) were identified in the transcriptome of Saponaria vaccaria (Sv) by performing a sequence homology search using the tblastn algorithm. The amino acid sequences of the MSBPs from Arabidopsis thaliana (referred to as "AtMSBP1" (SEQ ID NO: 63) and "AtMSBP2" (SEQ ID NO: 65)) were entered into a database (generated in-house) of the transcriptome of Saponaria vaccaria and compared with the DNA sequences translated from all genes in this transcriptome. Similar sequences were selected based on sequence identity (right-most column in Table 3) and significance of sequence match (third column in Table 3). The results are summarized in Table 3 below.

[0460] [Table 3]

[0461] The average expression levels of the various homologs identified in Table 3 were analyzed in leaves and flowers of Saponaria vaccaria (see Figure 10).

[0462] 1.2.5 Production of QA in yeast using the MSBP homologue from Saponaria vacccaria The functional relevance and activity of transcripts PB.393.1 and PB.16084.2 were tested for their ability to increase oxidation efficiency and improve QA production in yeast. The corresponding proteins were named "SvMSBP1" (SEQ ID NO:67, encoded by SEQ ID NO:69) and "SvMSBP2" (SEQ ID NO:70, encoded by SEQ ID NO:72), respectively, and were integrated into the genome of yeast engineered to produce QA as follows: ·AtATR1 / QsC28 / QsC23 / Qsb5 / QsC28C16 / SvMSBP1(YL-6) ·AtATR1 / QsC28 / QsC23 / Qsb5 / QsC28C16 / SvMSBP2 ·AtATR1 / QsC28 / QsC23 / Qsb5 / QsC28C16 / QsMSBP1 · AtATR1 / QsC28 / QsC23 / Qsb5 / QsC28C16 (YL-4) was used as a control.

[0463] Similarly, the MSBP homologue from Quillaja quinata (designated "QsMSBP1" - SEQ ID NO:73, encoded by SEQ ID NO:75) was also tested. The production of QA and QA precursors was analyzed by LC-MS (using commercial standards as reference). The results are shown in the graph in FIG. 11.

[0464] result Compared to YL-4 (not overexpressing MSBP proteins), a significant increase in QA production was observed in yeast overexpressing MSBP proteins (from Saponaria vaccaria or Quillaja), with SvMSBP1 and SvMSBP2 showing good results (see Figure 11). SvMSBP1 was selected and the yeast was further engineered to produce C3-glycosylated QA derivatives (see Example 2 below), C28-glycosylated QA derivatives (see Example 3 below), or QS-21-Xyl and QS-21-Api (see Example 4 below).

[0465] conclusion By using various combinations of heterologous enzymes (β-amyrin synthase, CYP oxidase, and CYP reductase) and heterologous proteins (cytochrome b5 and MSBP proteins) derived from various types of plants (e.g., Betula oleracea, Arabidopsis thaliana, Quillaja japonica, and Saponaria vaccaria), we reconstructed the metabolic pathway by which QA is biosynthesized in yeast and successfully produced QA at a level of approximately 65 mg / L in yeast for the first time.

[0466] Example 2 - Biosynthesis of C3-glycosylated QA derivatives 2.1 Production of UDP-sugars (glucuronic acid, xylose, rhamnose) not naturally occurring in yeast Glucuronic acid (GlcA) As shown in Figure 12, UDP-GlcA is produced from UDP-glucose by UDP-glucose dehydrogenase (UGD). The gene encoding UDP-glucose dehydrogenase from Arabidopsis thaliana (referred to as "AtUGD" - SEQ ID NO: 84 encoded by SEQ ID NO: 86) was integrated into the genome of the parent yeast strain CEN.PK2-1c to generate SC-1. The production of UDP-GlcA was analyzed by LC-MS.

[0467] result As shown in FIG. 13A, UDP-GlcA was detected in SC-1, confirming that AtUGD has functional activity when overexpressed in yeast.

[0468] Xylose (Xyl) As shown in FIG. 12, UDP-GlcA can be decarboxylated by UDP-xylose synthase (UXS) to form UDP-Xyl. UDP-xylose synthase from Arabidopsis thaliana (referred to as "AtUXS" - SEQ ID NO: 105 encoded by SEQ ID NO: 107) was integrated into the genome of SC-1 (yeast overexpressing AtUGD) to generate SC-4. Also, as shown in FIG. 12, UDP-GlcA can be decarboxylated by UDP-apiose synthase (AXS) to form UDP-Xyl. UDP-apiose synthase (AXS) from Quillaja japonica (referred to as "QsAXS" - SEQ ID NO: 113 encoded by SEQ ID NO: 115) was integrated into the genome of the parent yeast strain CEN.PK2-1c together with AtUGD to generate SC-16. The production of UDP-Xyl was analyzed by LC-MS.

[0469] result UDP-Xyl production was detected in both SC-4 and SC-16 (see Figures 13A and 13B), confirming that AtUXS and QsAXS have functional activity when overexpressed in yeast.

[0470] Rhamnose (Rha) As a candidate for rhamnose synthase, expression of the trifunctional AtRHM2 synthase from Arabidopsis thaliana (referred to as "AtRHM2" - SEQ ID NO:102 encoded by SEQ ID NO:104) was examined. AtRHM2 catalyzes the direct conversion of UDP-Glc to UDP-Rha via (i) dehydration of UDP-Glc, (ii) epimerization at the C3' and C5' positions to form UDP-4-keto-β-L-rhamnose, and (iii) reduction of UDP-4-keto-β-L-rhamnose to generate UDP-β-L-rhamnose. AtRHM2 was integrated into the genome of the parent yeast strain CEN.PK2-1c to generate SC-17, and AtRHM2 was integrated into the genome of SC-4 to generate SC-18. The production of UDP-Rha was analyzed by LC-MS.

[0471] result Production of UDP-Rha was detected in both SC-17 and SC-18 (see Figures 14A and 14B), confirming that ARHM2 has functional activity when overexpressed in yeast.

[0472] 2.2 Preparation of QA-C3-GlcA The AtUGD described above was integrated into the genome of YL-10 (producing QA) together with a glucuronyltransferase (GlcAT) from Quillaja japonica (referred to as "QsCslG1" - SEQ ID NO: 78 encoded by SEQ ID NO: 80) or a second glucuronyltransferase from Quillaja japonica (referred to as "QsCslG2" - SEQ ID NO: 81 encoded by SEQ ID NO: 83) to produce YL-11 and YL-12, respectively. The production of QA precursor and QA-C3-Glc was analyzed by LC-MS using each standard as a reference (the standard for QA-C3-GlcA corresponds to QAGlcpA produced as described in WO20 / 260475).

[0473] result QA-C3-GlcA was detected in both YL-11 (overexpressing QsCslG1) and YL-12 (overexpressing QsCslG2) (see Figures 15 and 16, respectively), confirming that these two enzymes have functional activity when overexpressed in yeast. QsCslG1 is specific for QA and does not glycosylate other precursors, whereas the QsCslG2 enzyme has promiscuous specificity and produces GlcA-QA with three-fold higher reactivity than the CslG1 enzyme (10.2 mg / L GlcA-QA produced by the QsCslG2 enzyme and 3.9 mg / L GlcA-QA produced by the CslG1 enzyme) (see FIG. 17).

[0474] Furthermore, a novel gene encoding a CslG homologue enzyme (termed "SvCslG" - SEQ ID NO:76 encoded by SEQ ID NO:77) was identified from the transcriptome of Saponaria vacccaria. The function of SvCslG as a candidate GlcA transferase was confirmed by in vitro enzyme analysis. QA (commercially available, e.g., from MedChemExpress) and UDP-GlcA were added directly to the reaction buffer along with a microsomal preparation prepared by plasmid expression of a yeast strain overexpressing SvCslG. The formation of QA and QA-C3-GlcA was analyzed by LC-MS.

[0475] result As shown in Figure 18, in the presence of UDP-GlcA, a peak corresponding to QA-C3-GlcA was observed, indicating that UDP-GlcA was transferred to the C3 position of QA by SvCslG, confirming its functional relevance and activity when expressed in yeast.

[0476] 2.3 Preparation of QA-C3-GlcA-Gal In yeast, UDP-galactose is naturally produced, so this glycosylation step does not require the addition of a sugar synthase. Galactose transferase from Quillaja quinolobia (called "QsGalT" - SEQ ID NO: 116 encoded by SEQ ID NO: 118) was integrated into the genome of YL-12 to generate YL-13. The production of QA-C3-GlcA and QA-C3-GlcA-Gal was analyzed by LC-MS (using the respective standards). The standard of QA-C3-GlcA corresponds to "QAGlcpA" and the standard of QA-C3-GlcA-Gal corresponds to "QA-GlcpA-Galp". Both standards were prepared as described in WO20 / 260475.

[0477] result As shown in Figure 19, overexpression of QsGalT could promote the glucuronidation step and further shift the reaction equilibrium toward glycosylation, presumably by completely converting QA-C3-GlcA to QA-C3-GlcA-Gal. In YL-13, 24.3 mg / L QA-C3-GlcA-Gal was produced.

[0478] Furthermore, a novel gene encoding a putative galactose transferase (termed "SvGalT" - SEQ ID NO:98 encoded by SEQ ID NO:99) was identified from the transcriptome of Saponaria vacccaria. The function of SvGalT as a galactose transferase was confirmed by transiently expressing SvCslG and SvGalT in N. benthamiana plants. N. benthamiana plants were infiltrated with Agrobacterium tumefaciens 2 days prior to loading with 40 μM QA (commercially available, e.g., from MedChemExpress). The production of QA-C3-GlcA and QA-C3-GlcA-Gal (using respective standards) was analyzed by LC-MS. The standard of QA-C3-GlcA corresponds to "QAGlcpA", and the standard of QA-C3-GlcA-Gal corresponds to "QA-GlcpA-Galp". Both standards were prepared according to the description in WO20 / 260475.

[0479] result As shown in Figure 20, when SvCslG and SvGalT were coexpressed, a peak corresponding to QA-GlcA-Gal was observed, indicating that UDP-Gal was transferred to QA-GlcA by SvGalT, confirming its functional relevance and activity.

[0480] 2.4 Generation of QA-C3-GlcA-Gal-Rha The AtRHM2 described above and rhamnose transferase from Quillaja japonica (referred to as "QsRhaT" - SEQ ID NO: 119, encoded by SEQ ID NO: 121) were integrated into the genome of YL-13 to generate YL-14. The production of QA-C3-GlcA, QA-C3-GlcA-Gal and QA-C3-GlcA-Gal-Rha (using respective standards) was analyzed by LC-MS. The standard of QA-C3-GlcA corresponds to "QAGlcpA", the standard of QA-C3-GlcA-Gal corresponds to "QA-GlcpA-Galp", and the standard of QA-C3-GlcA-Gal-Rha corresponds to "QA-GlcpA-Galp-Rhap". All standards were prepared as described in WO20 / 260475.

[0481] result As shown in Figure 21, co-expression of AtRHM2 and QsRhaT with AtUGD and QsGalT produced QA-C3-GlcA-Gal-Rha at a production level of 9.5 mg / L. No residual QA-GlcA-Gal was observed, indicating that QsRhaT was highly efficient and that QA-GlcA-Gal was completely converted to QA-C3-GlcA-Gal-Rha by its catalytic activity.

[0482] 2.5 Preparation of QA-C3-GlcA-Gal-Xyl The AtUXS described above was integrated into the genome of YL-12, a yeast strain engineered to produce UDP-GlcA. Direct expression of AtUXS in this UDP-GlcA-producing yeast strain did not result in the production of any glycosylated molecules (data not shown), presumably due to insufficient production of UDP-GlcA. This result suggested that the downstream metabolite UDP-xylose may act as an allosteric feedback inhibitor to control the activity of UGD. This inhibitory effect is confirmed by the results shown in Figure 13A, where no UDP-GlcA was detected when AtUXS was coexpressed with AtUGD.

[0483] AtUGD mutations It has been reported that the introduction of the point mutation A104L into the human UGD homolog reduces the binding affinity of UDP-Xyl. Based on this, in order to alleviate the inhibition of UGD by UDP-Xyl observed in the above experiment, we reduced the binding affinity of UDP-Xyl by introducing a mutation into AtUGD. By aligning the protein sequence of AtUGD to that of human UGD, the corresponding amino acid was identified (data not shown), and the point mutation A101L was introduced into AtUGD (AtUGD A101L - SEQ ID NO: 108 encoded by SEQ ID NO: 109). The above QsCslG2 and QsGalT, together with UDP-xylose transferase from Quillaja japonica (QsC3XylT - SEQ ID NO: 122 encoded by SEQ ID NO: 124), A101L was integrated into the genome of YL-10 (yeast engineered to produce QA) to generate YL-15. The production of QA-C3-GlcA, QA-C3-GlcA-Gal, and QA-C3-GlcA-Gal-Xyl (using respective standards) was analyzed by LC-MS.

[0484] result As shown in FIG. 22, QA-C3-GlcA-Gal-Xyl was detected in YL-15, and the production amount was 1 mg / L.

[0485] To investigate the difference in the inhibitory effect of UDP-Xyl on various UGDs, six homologs were selected from various kingdoms of life. Specifically, UGD from Synechococcus sp. (Syn) (referred to as "SynUGD" - SEQ ID NO: 154 encoded by SEQ ID NO: 156), UGD from Homo sapiens (Hs) (referred to as "HsUGD"), A104LThe following UGDs were selected: a UGD from Paramoeba atlantica (Patl) (referred to as "PatlUGD" - SEQ ID NO:157 encoded by SEQ ID NO:159), a UGD from Bacillus cytotoxicus (Bcyt) (referred to as "BcytUGD" - SEQ ID NO:160 encoded by SEQ ID NO:162), a UGD from Corallococcus macrosporus (Myxfulv) (referred to as "MyxfulvUGD" - SEQ ID NO:163 encoded by SEQ ID NO:165), and a UGD from Pyrococcus furiosus (Pfu) (referred to as "PfuUGD" - SEQ ID NO:166 encoded by SEQ ID NO:168). These homologue sequences, together with QsCslG2, QsGalT, AtUXS and QsC3XylT, were integrated into the genome of YL-10 (a yeast strain engineered to produce QA) to generate YL-16 to YL-21, respectively. The production of QA-C3-GlcA-Gal-Xyl was analyzed by LC-MS (using each standard). The results are shown in the graph in Figure 23.

[0486] result As shown in FIG. 23, the production of QA-C3-GlcA-Gal-Xyl from UGD enzymes other than PatlUGD (YL-18) was suppressed by the AtUGD A101L The yield of the enzyme was comparable to that of the control enzyme YL-15, whereas PatlUGD (YL-18) yielded three times the yield of these enzymes. Alignment of the PatlUGD and AtUGD sequences revealed that the A101L mutation in AtUGD is naturally present in PatlUGD, suggesting that this mutation enhances the tolerance to UDP-Xyl (data not shown).

[0487] Alternative UDP-GlcA biosynthetic pathway UDP-GlcA can also be produced via the de novo salvage pathway or the myo-inositol oxidation pathway. Glucuronokinase (GlcAK) and UDP-sugar pyrophosphorylase (USP) convert free glucuronic acid to GlcA-1-phosphate and finally to the active UDP form of GlcA (UDP-GlcA). These enzymes are also responsible for the myo-inositol pathway, which starts with myo-inositol oxygenase (referred to as "MIOX"). The GlcAK enzyme from Arabidopsis thaliana (referred to as "AtGlcAK" - SEQ ID NO:169, encoded by SEQ ID NO:171) and the USP from Arabidopsis thaliana (referred to as "AtUSP" - SEQ ID NO:223, encoded by SEQ ID NO:225) were integrated into the genome of YL-15 to generate YL-22. Separately, the above GlcAK and AtUSP were integrated into the genome of YL-15 together with MIOX from Thermothelomyces thermophilus (referred to as "TtMIOX" - SEQ ID NO:172 encoded by SEQ ID NO:174) to generate YL-23. The culture medium of YL-23 was either untreated or exogenously supplemented with 0.5% glucuronic acid and 2% myo-inositol (MI). The production of QA-C3-GGX (using respective standards) was analyzed by LC-MS.

[0488] result Residual QA was significantly reduced, resulting in a three-fold increase in the production of QA-C3-GGX in YL-22 (see FIG. 24). The production of QA-C3-GGX in YL-23 (which also overexpresses TtMIOX) was increased 1.7-fold in the presence of exogenously added 2% myo-inositol and 2.3-fold in the presence of exogenously added 0.5% GlcA. The addition of both myo-inositol and GlcA enhanced the production of QA-C3-GGX by 5.9-fold (see FIG. 25).

[0489] Inducible TetOn promoter to delay UXS expression and accumulate UDP-GlcA As a method to delay the expression of AtUXS, inducible promoters such as pDDI2 promoter (induced by methyl methanesulfonate), pCup1 promoter (induced by copper ions), and pTetOn promoter (induced by tetracycline or doxycycline) were investigated and used. AtUXS was overexpressed under the control of the pTetOn promoter in yeast engineered to produce QA-C3-GGX. The production of QA, QA-C3-GG, and QA-C3-GGX was analyzed by LC-MS.

[0490] result The pTetOn promoter showed expression comparable to that of the galactose promoter used in the parent yeast strain, and the expression level of the AtUXS protein increased linearly depending on the inducer concentration (data not shown). In the absence of inducer, a 5.5-fold increase in the production of QA-C3-GGX was observed, which was probably due to basal expression of AtUXS due to promoter leakage. The production of small amounts of UDP-Xyl may not result in inhibition of AtUGD. To induce the pTetOn promoter, 20 μg / mL or 100 μg / mL doxycycline was exogenously added to the yeast culture medium 24 hours after induction with galactose. Addition of doxycycline increased the production of QA-C3-GGX by 5.9-fold or 8.5-fold compared to YL-15. An 11-fold increase was observed when the pTetOn promoter was induced with 100 μg / mL doxycycline 40 hours after induction with galactose (see FIG. 26).

[0491] Identification of the C3XylT enzyme in Saponaria vacccaria Furthermore, a novel gene encoding a putative xylosyltransferase (termed "SvC3XylT" - SEQ ID NO:100 encoded by SEQ ID NO:101) was identified in the transcriptome of Saponaria vaccaria. The function of SvC3XylT as a xylosyltransferase was tested by transiently co-expressing the previously described SvCslG and SvGalT enzymes in N. benthamiana plants. Two days after infiltration with Agrobacterium tumefaciens, N. benthamiana plants were loaded with 40 μM QA (commercially available, for example, from MedChemExpress). The production of QA-C3-GlcA-Gal-Xyl was analyzed by LC-MS. A standard equivalent to "QA-GlcpA-Galp-Xylp", prepared as described in WO20 / 260475, was used as a reference.

[0492] result As shown in Figure 58, when SvCslG, SvGalT and SvC3XylT were coexpressed, a peak corresponding to QA-C3-GlcA-Gal-Xyl was observed, indicating that UDP-Xyl was transferred to QA-GlcA-Gal by SvXylT, confirming its functional relevance and activity.

[0493] conclusion By using various heterologous enzymes (glycosyl synthesis enzymes and glycosyltransferases) derived from various kinds of plants (e.g., Arabidopsis thaliana, Quillaja japonica, or Saponaria vaccaria) in various combinations, we reconstructed the metabolic pathway for the biosynthesis of C3-glycosylated QA derivatives in yeast and successfully produced C3-glycosylated QA derivatives in yeast for the first time.

[0494] Example 3 - Biosynthesis of C28-glycosylated QA derivatives 3.1 Generation of fucose not naturally occurring in yeast Because Saponaria vacccaria contains a variety of saponins with similarity to those found in Quillaja, we further investigated the transcriptome of Saponaria vacccaria to identify genes and enzymes involved in saponin biosynthesis. We treated Saponaria vacccaria plants with methyl jasmonate (Meja), which has previously been shown to induce saponin biosynthesis in plants. We then performed extensive RNASeq analysis to identify full-length transcripts in the plants and identify the induced genes. The identified genes included several genes known to be involved in the biosynthesis of the triterpene backbone (e.g., β-amyrin synthase), as well as several cytochrome P450 enzyme (CYP) genes and glycosyltransferase genes (see, e.g., WO20 / 263524). Also, some of the identified genes are homologs of genes known to be involved in the biosynthesis of saponins in Quillaja (see, for example, WO19 / 122259, WO20 / 260475, WO22 / 136563; Decker and Kleczkowski (2017)). Based on knowledge of the biosynthesis of dTDP-D-fucose in bacteria and UDP-L-rhamnose in plants, the saponin biosynthetic pathway was predicted to include a dehydratase step and a reductase step (shown in Figure 12). No homologs of enzymes involved in the biosynthesis of dTDP-D-fucose were found in bacteria. A homolog of the UDP-4-keto-6-deoxyglucose reductase gene from Quillaja was found in the transcriptome of Saponaria vaccaria (this homolog is called "svNMD"). A candidate UDP-glucose-4,6-dehydratase induced by methyl jasmonate was also found, which belongs to the nucleotide sugar epimerase family. This enzyme was named "svUG46DH". This predicted enzyme had similarity to the plant UDP-L-rhamnose synthase domain. We hypothesized that these two enzymes, sv46DH and svNMD, could catalyze the conversion of UDP-D-glucose to UDP-D-fucose (see Figure 12).The functional relevance and activity of these newly identified genes were tested in yeast in the following combinations to assess their ability to produce UDP-fucose: Both svUG46DH (SEQ ID NO:87 encoded by SEQ ID NO:89) and svNMD (SEQ ID NO:90 encoded by SEQ ID NO:92) were integrated into the genome of the parent yeast strain CEN.PK2-1c to generate SC-19. Both SvUG46DH and SvNMD were integrated into the genome of SC-4 (overexpressing AtUGD-AtUXS) to generate SC-20. Both SvUG46DH and SvNMD were integrated into the genome of SC-17 (overexpressing AtRHM2) to generate SC-22. · Both SvUG46DH and SvNMD were integrated into the genome of SC-18 (overexpressing AtUGD-AtUXS-AtRHM2) to generate SC-23.

[0495] Separately, a homologue reductase from Quillaja japonica (WO22 / 136563) (designated "QsFucSyn" - SEQ ID NO:175 encoded by SEQ ID NO:177) was tested in the following combinations: ·QsFucSyn and SvUG46DH were integrated into the genome of SC-4 (overexpressing AtUGD-AtUXS) to generate SC-21. The production of UDP-fucose was analyzed by LC-MS.

[0496] result When only the combination of svUG46DH and svNMD was overexpressed (SC-19), UDP-fucose was produced (see Figures 14A and 14B or Figures 27A and 27B). When svUG46DH and svNMD were overexpressed with AtUGD-AtUXS (SC-20), UDP-fucose was also produced (see Figures 14A and 14B or Figures 27A and 27B). When svUG46DH and svNMD were overexpressed with AtRHM2 (SC-22), UDP-fucose was also produced (see Figures 14A and 14B). When svUG46DH and svNMD were overexpressed with AtUGD-AtUXS-AtRHM2 (SC-23), UDP-fucose was also produced (see Figures 14A and 14B). When QsFucSyn and svUG46DH were overexpressed with AtUGD-AtUXS (SC-21), UDP-fucose was also produced (see Figures 14A and 14B or Figure 27A). These results confirmed the functional association and activity of the newly identified SvUG46DH and SvNMD with QsFucSyn when expressed in yeast.

[0497] 3.2 Preparation of QA-C3-GlcA-Gal-Rha / Xyl-C28-Fuc Fucosyltransferase from Quillaja japonica (WO22 / 136563) (referred to as "QsFucT" - SEQ ID NO:93 encoded by SEQ ID NO:95) was integrated into the genome of YL-14 to generate YL-25.

[0498] result QA-C3-GlcA-Gal-Rha and QA-C3-GlcA-Gal-Rha-C28-Fuc were detected in YL-25 by coelution with the respective standards (see FIG. 28). (QA-C3-GlcA-Gal-Rha standard corresponds to "QA-TriR" prepared as described in WO 22 / 136563, and QA-C3-GlcA-Gal-Rha-C28-Fuc standard corresponds to "QA-TriR-F" prepared as described in WO 22 / 136563.)

[0499] The above QsFucT enzyme was integrated into the genome of YL-15 to generate YL-26. The production of QA-C3-GlcA-Gal-Rha-C28-Fuc was analyzed by LC-MS.

[0500] result As above, production of QA-C3-GlcA-Gal-Xyl-C28-Fuc was observed in YL-26 (data not shown).

[0501] Identification of the FucT enzyme in Saponaria vacccaria Furthermore, a novel gene encoding a FucT candidate (termed "SvFucT" - SEQ ID NO: 96 encoded by SEQ ID NO: 97) was identified from the transcriptome of Saponaria vacccaria. The function of SvFucT as a fucose transferase was tested by transiently co-expressing the previously described SvCslG, SvUG46DH and SvNMD in N. benthamiana plants. Two days after infiltration with Agrobacterium tumefaciens, N. benthamiana plants were loaded with 40 μM QA (commercially available, for example, from MedChemExpress). QsFucT (see above) was used as a positive control and GFP was used as a negative control. The production of QA-C3-GlcA-C28-Fuc was analyzed by LC-MS.

[0502] result As shown in Figure 57, when SvFucT was overexpressed, a peak was observed at the same retention time as when QsFucT (positive control) was overexpressed (see dashed line), indicating that UDP-Fuc was transferred to QA-GlcA by SvFucT, confirming the functional relevance and activity of the newly identified SvFucT.

[0503] 3.3 Preparation of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha The trifunctional AtRHM2 enzyme described above was integrated into the genome of YL-15 together with the rhamnose transferase from Quillaja japonica (designated "QsRhaT" - SEQ ID NO:119 encoded by SEQ ID NO:121) to generate YL-28. The production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha (using a chemically synthesized standard as a reference) was analyzed by LC-MS.

[0504] result The production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha was detected in YL-28 at a titer of approximately 1 mg / L (see FIG. 29). No residual substrate was observed, indicating that QsRhaT was highly efficient and catalyzed the complete conversion of QA-C3-GlcA-Gal-Xyl-C28-Fuc to QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha.

[0505] 3.4 Generation of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha An additional copy of the trifunctional AtRHM2 enzyme (described above) was integrated into the genome of YL-14 together with QsRhaT (described above) to generate YL-27. The production of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha was analyzed by LC-MS. A standard corresponding to "QA-TriR-FR", prepared as described in WO22 / 136563, was used as a reference.

[0506] result QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha was detected in YL-27 at a titer of approximately 3 mg / L (see FIG. 30). No residual substrate was observed, indicating that QsRhaT was highly efficient and catalyzed the complete conversion of QA-C3-GlcA-Gal-Rha-C28-Fuc to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha.

[0507] 3.5 Preparation of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl An additional copy of the AtUXS enzyme (described above) was integrated into the genome of YL-28 together with the xylose transferase from Quillaja japonica (referred to as "QsC28XylT3" - SEQ ID NO: 125 encoded by SEQ ID NO: 127) to generate YL-30. The production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl was analyzed by LC-MS. QA-C3-GGR-C28-FRX obtained in the previous experiment was used as a reference.

[0508] result In YL-30, QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl was detected (see Figure 31).

[0509] 3.6 Preparation of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl The AtUXS enzyme (described above) was integrated into the genome of YL-27 together with QsC28XylT3 described above to generate YL-29. The formation of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl was analyzed by LC-MS. A standard corresponding to "QA-TriR-FRX", prepared as described in WO22 / 136563, was used as a reference.

[0510] result In YL-29, QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl was detected (see Figure 32).

[0511] 3.7 Production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl An additional copy of the AtUXS enzyme (described above) was integrated into the genome of YL-30 together with the xylose transferase from Quillaja japonica (referred to as "QsC28XylT4" - SEQ ID NO:128 encoded by SEQ ID NO:130) to generate YL-33. The production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl was analyzed by LC-MS. QA-C3-GGR-C28-FRXX obtained in the previous experiment was used as a reference.

[0512] result In YL-33, conversion of QA-C3-GGX-C28-FRX to QA-C3-GGX-C28-FRX was observed (Figure 33).

[0513] 3.8 Production of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl An additional copy of the AtUXS enzyme (described above) was integrated into the genome of YL-29 together with the previously described C28QsXylT4 to generate YL-31. The production of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl was analyzed by LC-MS. A standard corresponding to "QA-TriR-FRXX", prepared as described in WO22 / 136563, was used as a reference.

[0514] result In YL-31, conversion of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xy to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl was observed (Figure 34).

[0515] 3.9 Formation of apiose, which does not naturally occur in yeast UDP-apiose can be produced using apiose synthase ("AXS"), which produces both UDP-Xyl and UDP-Api (as shown in FIG. 12). The AtUGD enzyme (described above) and the apiose synthase from Quillaja quinata (referred to as "QsAXS" - SEQ ID NO:113 encoded by SEQ ID NO:115) were integrated into the genome of the parent yeast strain CEN.PK2-1c to generate SC-16. UDP-apiose is a highly unstable compound with a half-life of 100 minutes at room temperature. UDP-apiose was not detectable (data not shown), presumably because it was produced but degraded during the extraction process and therefore could not be detected by LC-MS.

[0516] 3.10 Generation of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api An additional copy of the QsAXS enzyme described above was integrated into the genome of YL-30 along with the apiose transferase from Quillaja quilla (designated "QsC28ApiT4" - SEQ ID NO:151 encoded by SEQ ID NO:153) to generate YL-34. The production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api was analyzed by LC-MS.

[0517] result In YL-34, conversion of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl to QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api was observed (Figure 35).

[0518] 3.11 Generation of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api The aforementioned QsAXS enzyme was integrated into the genome of YL-29 together with the aforementioned QsC28ApiT4 enzyme to generate YL-32. The production of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api was analyzed by LC-MS.

[0519] result In YL-32, conversion of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api was observed (Figure 36).

[0520] conclusion By using various heterologous enzymes (glycosyl synthases and glycosyltransferases) derived from various kinds of plants (e.g., Arabidopsis thaliana, Quillaja japonica, and Saponaria vaccaria), we reconstructed the metabolic pathway for the biosynthesis of C28-glycosylated QA derivatives in yeast and successfully produced C28-glycosylated QA derivatives in yeast for the first time.

[0521] We assessed whether an alternative method could be utilized to improve the conversion of QA-C3-GGR / X-C28-FRX to QA-C3-GGR / X-FRXX / A.

[0522] 3.12 Subcellular localization of QsC28XylT4 and QsC28ApiT4 The subcellular localization of QsC28XylT4 and QsC28ApiT4 heterologously expressed in yeast was examined. To visualize the subcellular localization in yeast (see QsC28XylT3-GFP), QsC28XylT4-GFP and QsC28ApiT4-GFP were constructed by fusion with green fluorescent protein (GFP) at the C-terminus. QsC28XylT4-GFP, QsC28ApiT4-GFP, and QsC28XylT3-GFP were each separately integrated into the genome of the parent yeast strain CEN.PK2-1c.

[0523] result Flow cytometry data (to measure absolute protein expression) showed similar fluorescence intensities for both enzymes, indicating similar protein expression levels (data not shown). Confocal microscopy images also showed that QsC28XylT3-GFP was localized in the cytosol (Figure 37A), whereas QsC28XylT4-GFP (Figure 37B) and QsC28ApiT4-GFP (Figure 37C) formed aggregates commonly known as inclusion bodies in yeast.

[0524] 3.13 Identification of the localization of QsXylT4 and QsApiT4 aggregates To functionally express the two enzymes, QsC28XylT4 and QsC28ApiT4, in the cytosol, three localization protein signature markers were selected to identify the subcellular localizations where QsC28XylT4 and QsC28ApiT4 aggregates form. The first protein selected, Rnq1, is a naturally occurring prion protein in yeast that has previously been shown to colocalize with insoluble protein deposits (IPODs), the site of amyloid-like protein accumulation and degradation. By expressing C-terminal mCherry-tagged Rnq1 in yeast, we were able to visualize only IPODs as perivascular compartments (data not shown). Coexpression of Rnq1-mCherry and QsC28XylT4-GFP showed distinct localization patterns, revealing that QsC28XylT4 is not an amyloid-like misfolded protein (data not shown). As the second protein marker, we selected heat shock protein 42 (Hsp-42). Hsp-42 was selected because of its suggested physiological role in forming stress granules in yeast when carbon and nitrogen sources are depleted. Hsp42-mCherry fusion protein was localized in the yeast cytosol and nucleus (not shown) and was shown to colocalize with QsXylT4-GFP (data not shown), suggesting that QsC28XylT4 may be sequestered in stress granules. As the third protein marker, we selected Rpn1. Rpn1 is a functional component of the proteasome that is actively involved in the protein degradation machinery. When Rpn1 was expressed alone, it was localized in the nucleus together with the proteasome machinery. Furthermore, when coexpressed with QsC28XylT4-GFP, most of Rpn1-mCherry remained in the nucleus after 24 h (data not shown), but at 48 h it aggregated around the QsC28XylT4-GFP aggregates, disassembling the QsC28XylT4-GFP aggregates and recycling the protein (data not shown). These results suggest that QsC28XylT4 is likely sequestered in Hsp42-associated stress granules and is susceptible to degradation.

[0525] 3.14 N-terminal truncation or addition of soluble tags to QsC28XylT4 To localize QsC28XylT4 in the cytosol, we performed experiments in which we cut amino acids from the N-terminus of QsC28XylT4 every third residue, up to a maximum of 12 residues, and experiments in which we added soluble tags such as SUMO, TrXA, and MBP. Specifically, we performed experiments in which we deleted the first three amino acids from QsC28XylT4, i.e., "QsC28XylT4-3aa" (QsC28XylT4 with the first three amino acids deleted - SEQ ID NO: 131 encoded by SEQ ID NO: 133), "QsC28XylT4-6aa" (QsC28XylT4 with the first six amino acids deleted - SEQ ID NO: 134 encoded by SEQ ID NO: 136), "QsC28XylT4-9aa" (QsC28XylT4 with the first nine amino acids deleted - SEQ ID NO: 137 encoded by SEQ ID NO: 139), and "QsC28XylT4-12aa" (QsC28 The first 12 amino acid residues of XylT4 were deleted (SEQ ID NO:140, encoded by SEQ ID NO:142), "SUMO-QsC28XylT4" (SEQ ID NO:143, encoded by SEQ ID NO:144), "TrXA-QsC28XylT4" (SEQ ID NO:145, encoded by SEQ ID NO:146), or "MBP-QsC28XylT4" (SEQ ID NO:147, encoded by SEQ ID NO:148) were integrated into the genome of YL-30 to generate YL-35, YL-36, YL-37, YL-38, YL-39, and YL-40, respectively. The protein expression level of QsC28XylT4 in each yeast strain (compared to YL-33, which has the full length of wild-type QsXylT4 without tagging) and the ability of each yeast strain to produce QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl were examined.

[0526] result Fluorescence intensity measured by flow cytometry showed the highest protein expression in QsC28XylT4-MBP (see FIG. 38). All N-terminally truncated and N-terminally tagged QsC28XylT4s showed good yields of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, and among them, the N-terminally MBP-tagged QsC28XylT4 showed a 7-fold increase in yield compared to the wild-type full-length enzyme (see Figure 39).

[0527] 3.15 Fusion of QsXylT3 to the N-terminus of QsXylT4 As an alternative method to localize QsC28XylT4 to the cytosol, QsC28XylT3 (shown above to be localized to the cytosol when expressed in yeast) was fused to the N-terminus of QsC28XylT4. A 3xGGGS linker was genetically inserted between the amino acid sequences of these enzymes to allow flexibility of the linker and allow the two enzymes to fold separately without affecting the functional properties of the resulting fusion protein. This QsC28XylT3-3xGGGS-QsC28XylT4 fusion (SEQ ID NO:149, encoded by SEQ ID NO:150) was integrated into the genome of YL-30 to generate YL-41. The localization of this fusion protein and the production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl were investigated.

[0528] result Confocal microscopy images showed that the QsXylT3-3xGGGS-QsXylT4-GFP fusion protein showed less aggregation and enhanced cytosolic expression compared to QsXylT4-GFP expressed alone (see Figure 40). The complete conversion of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl gave a characteristic peak corresponding to the mass of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl (see Figure 41B), whereas no such peak was observed in the yeast strains expressing QsXylT3 and QsXylT4 separately (see Figure 41A), confirming the improved reactivity of the fusion protein.

[0529] 3.16 Continuous fed-batch scheme to inhibit sequestration and degradation of QsXylT4 A continuous fed-batch scheme was established by adding fresh nitrogen and / or carbon sources every 24 h. Protein expression and localization were investigated.

[0530] result The fluorescence intensity, which reflects the absolute expression level of QsC28XylT4, was measured by flow cytometry (Figure 42). In a 60-hour experiment, the protein expression level decreased over time, and addition of only a carbon source (glucose or galactose) had no effect on the expression level. In contrast, the addition of fresh medium supplemented with a nitrogen source and 4% galactose consistently increased the expression level of QsC28XylT4, reaching a fivefold increase at 60 h. Upon fresh addition of carbon and nitrogen sources, QsXylT4 did not colocalize with Hsp42. Rpn1, which indicates localization of the proteasome machinery, remained in the nucleus and did not degrade QsXylT4 aggregates. Although some QsXylT4 aggregates were observed in the presence of fresh medium, cytosolic expression was consistently observed, in contrast to the disappearance of cytosolic QsXylT4 expression after 24 h in yeast strains grown only in old medium (data not shown).

[0531] Example 4 - Terminally modified 18-carbon pseudodimeric acyl chain in the biosynthesis of Araf As shown in Figure 2A, acylation requires the biosynthesis of C9-CoA and the sequential addition of two resulting C9-CoAs. Two units of malonyl-CoA and one unit of (S)-2-methylbutyryl-CoA (2MB-CoA) are condensed to form C9-keto-CoA by two units of chalcone synthase-like type III polyketide synthase (PKS), which is then reduced to C9-CoA by two independent units of ketoreductase (KR).

[0532] 4.1 Conversion of 2-methylbutyrate to (S)-2-methylbutyryl CoA (2MB-CoA) The conversion of exogenously added 2-methylbutyrate to 2MB-CoA by the CoA ligase identified from the transcriptome of Quillaja japonica was examined. Functional expression of this CoA ligase from Quillaja japonica (referred to as "QsCCL" - SEQ ID NO:178 encoded by SEQ ID NO:180) was confirmed by transfecting a high copy plasmid into the parent yeast strain CEN.pk2-1c and observing GFP fused to the C-terminus of the enzyme in confocal microscopy images. GFP fused to the C-terminus was visualized and found to be localized in the cytoplasm and was shown to be stable for at least 24 hours after induction with galactose (data not shown). Furthermore, a whole-cell fed-batch experiment was performed to convert 2-methylbutyrate to 2MB-CoA by QsCCL. Specifically, 2-methylbutyrate was added directly to the yeast cell medium, yeast cells were lysed, and the intracellular 2MB-CoA content was measured by liquid chromatography using a porous graphitic carbon column. By coeluting with a 2MB-CoA standard, it was confirmed that 2MB-CoA was produced from 50 mg / L of 2-methylbutyrate in YL-QsCCL (the standard was chemically synthesized) (see Figure 43).

[0533] 4.2 Biosynthesis of keto-C9-CoA and its reduction to produce C9-CoA As shown in Figures 2A and 2E, the 18-carbon pseudodimeric acyl chain consists of two repeating C9 units. The 18-carbon pseudodimeric acyl chain is synthesized from two units of malonyl-CoA and one unit of 2MB-CoA using two chalcone synthase-like type III PKSs (referred to as "QsChSD" (SEQ ID NO:181, encoded by SEQ ID NO:183) and "QsChSE" (SEQ ID NO:184, encoded by SEQ ID NO:186). The product C9-keto-CoA is then reduced to form C9-CoA by two ketoreductases (referred to as "QsKR11" (SEQ ID NO:187, encoded by SEQ ID NO:189) and "QsKR23" (SEQ ID NO:190, encoded by SEQ ID NO:192). Functional expression of these four enzymes was confirmed by using high copy plasmids in yeast and observing confocal microscopy images of GFP fused to the C-terminus of each enzyme. Expression of both QsChSD and QsChSE was shown to be localized in the cytosol, with QsChSE showing less aggregation. Expression of KR11 was localized in the cytoplasm, whereas expression of QsKR23 was shown to be localized to the membrane of the endoplasmic reticulum (ER) (data not shown).

[0534] 4.3 Addition of C9-CoA to C3- and C28-glycosylated QA derivatives The formation of C9-CoA itself could not be detected directly by LC-MS, probably due to its short stability. Based on this, C9-CoA was added to glycosylated QA derivatives to synthesize C9-CoA. It was shown that the acyl unit (C9-CoA) could be added to both QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl (QA-C3-GGX-C28-FRX) and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl (QA-C3-GGX-C28-FRXX) (Figure 44). Because the LC-MS response was higher for QA-C3-GGX-C28-FRX, a large cassette containing QsChsD and QsChSE, QsKR11 and QsKR23, QsCCL and an acyltransferase (referred to as "QsDMOT9" - SEQ ID NO:193 encoded by nucleotide sequence SEQ ID NO:195) was engineered into the genome of a yeast strain (YL-30) that produces such glycosylated QA derivatives, generating YL-42. The production of QA-C3-GGX-C28-FRX-C9 was analyzed by LC-MS.

[0535] result QA-C3-GGX-C28-FRX-C9 co-eluted from YL-42 with the standard, indicating that YL-42 produces QA-C3-GGX-C28-FRX-C9 in the presence of 50 mg / L of exogenously added 2-methylbutyric acid (standards produced in N. benthamiana as described in GB 2204252.7) (see FIG. 44). Addition of high concentrations of 2-methylbutyric acid to the growth medium improved the conversion of QA-C3-GGX-C28-FRX to QA-C3-GGX-C28-FRX-C9 (Figure 45).

[0536] The 18-carbon pseudodimeric acyl chain consists of two repeating C9-CoA units, and the addition of the second C9-CoA unit requires the corresponding acyltransferase (called "QsDMOT4" - SEQ ID NO: 196, encoded by the nucleotide sequence shown in SEQ ID NO: 198), which was integrated into the genome of YL-42 to generate YL-43.

[0537] result Addition of 500 mg / L 2-methylbutyrate to the culture medium resulted in the appearance of a new LC-MS peak with the same high-resolution mass, confirming the formation of QA-C3-GGX-C28-FRX-C18, and indicated that the conversion of QA-C3-GGX-C28-FRX-C9 to QA-C3-GGX-C28-FRX-C18 was highly efficient with little residual substrate (Figure 46), indicating that the addition of 2-methylbutyrate and the endogenous malonyl-CoA pool provided sufficient C9-CoA for the addition of two acyl units.

[0538] To generate QA-C3-GGX-C28-FRXX-C18, QsDMTO4 and QsC28XylT4 were integrated into the genome of YL-42 to generate YL-44. The formation of QA-C3-GGX-C28-FRXX-C18 was analyzed by LC-MS.

[0539] result A new LC-MS peak was detected corresponding to the mass of QA-C3-GGX-C28-FRXX-C18 (Figure 47). The absence of QA-C3-GGX-C28-FRXX and QA-C3-GGX-C28-FRXX-C9 suggested that they are better substrates for QsDMOT9 and QSDMOT4 acyltransferases than QA-C3-GGX-C28-FRX and QA-C3-GGX-C28-FRX-C9.

[0540] 4.4 Generation of UDP-arabinofuranose (Araf), which does not naturally occur in yeast Since the biosynthesis of UDP-Araf does not occur naturally in yeast, the heterologous production and addition of UDP-Araf requires nucleotide sugar synthases and arabinosyltransferases. As shown in Figure 12, UDP-Xyl is first converted to UDP-arabinopyranose by UDP-Xyl epimerase (UXE), which can then undergo ring-chain tautomerization with the assistance of UDP-arabinose mutase (UAM). UAM from Arabidopsis thaliana ("AtUAM1" - SEQ ID NO:208 encoded by the nucleotide sequence set forth in SEQ ID NO:210), UAM from barley (H. vulgare) ("HvUAM" - SEQ ID NO:211 encoded by the nucleotide sequence set forth in SEQ ID NO:213), UXE from Arabidopsis thaliana ("AtUXE" - SEQ ID NO:199 encoded by the nucleotide sequence set forth in SEQ ID NO:201), "AtUXE2" (SEQ ID NO:202 encoded by the nucleotide sequence set forth in SEQ ID NO:204) and / or "AtUGE3" (SEQ ID NO:205 encoded by the nucleotide sequence set forth in SEQ ID NO:207) were incorporated into SC-4 in the following combinations: AtUAM1-AtUXE2 (SC-9) HvUAM-AtUXE (SC-10) AtUAM1-AtUGE3 (SC-11)

[0541] Sugar production was analyzed by LC-MS.

[0542] result UDP-Xyl was produced in all enzyme combinations, and the amount of UDP-Xyl produced was low in the AtUAM1-AtUGE3 combination (SC-11). The production of UDP-Arap was comparable in both combinations, but UDP-Araf was not detected and was probably coeluted with UDP-Xyl. Because the UXE and UAM enzymes are in equilibrium, the abundance of UDP-Araf is thought to be 100-fold lower than that of UDP-Xyl (see Figure 48B).

[0543] Alternatively, the salvage pathway was tested using arabinokinase (AraK) and UDP-sugar pyrophosphorylase (USP) candidates from Arabidopsis thaliana (designated "AtAraK" - SEQ ID NO:214, encoded by the nucleotide sequence set forth in SEQ ID NO:216; designated "AtUSP" - SEQ ID NO:223, encoded by the nucleotide sequence set forth in SEQ ID NO:225) and arabinokinase (AraK) and UDP-sugar pyrophosphorylase (USP) candidates from pathogenic Leptospira interrogans (Lei) (designated "LeiAraK" - SEQ ID NO:217, encoded by the nucleotide sequence set forth in SEQ ID NO:219; "LeiUSP" - SEQ ID NO:226, encoded by the nucleotide sequence set forth in SEQ ID NO:228). Additionally, the arabinose transporter from Penicillium rubens strain Wisconsin (referred to as "PrAraT" - encoded by the nucleotide sequence shown in SEQ ID NO:222) was also tested to determine whether arabinose must be imported into yeast and AtUAM1 for UDP-Arap to be converted to UDP-Araf. The following combinations were integrated into the genome of the parent yeast strain CEN.PK2-1c and 1% arabinose was exogenously added to each yeast. AtAraK-AtUSP(SC-12) ·LeiAraK-LeiUSP(SC-13) AtAraK-AtUSP-PrAraT (SC-14) ·AtAraK-AtUSP-PrAraT-AtUAM1(SC-15)

[0544] result Both AraT and the salvage pathway derived from pathogenic Leptospira produced less UDP-Arap (0.910 μmol / g cell pellet and 0.665 μmol / g cell pellet, respectively) compared with the salvage pathway derived from Arabidopsis (1.73 μmol / g cell pellet). The UDP-Araf produced from the salvage pathway from Arabidopsis, AraT and AtUAM1, was 0.185 μmol / g cell pellet (see FIG. 48A).

[0545] 4.5 Addition of UDP-arabinofuranose (Araf) Since L-Araf is an important component of plant cell walls, the biosynthesis of plant-derived UDP-L-arabinofuranose (UDP-Araf) is closely related to the Golgi apparatus. UDP-Arap is mainly synthesized by epimerization of UDP-Xyl in the lumen of the Golgi apparatus, and UDP-Arap is interconverted to UDP-Araf by UDP-Ara mutase present on the cytoplasmic membrane surface of the Golgi apparatus, and then transported back to the lumen of the Golgi apparatus for glycosylation. Since the Golgi membrane does not contain sugar transporters naturally occurring in yeast, cytoplasmic homologs of these enzymes were selected from Arabidopsis thaliana (UDP-xylose epimerase (AtUXE) and AtUAM1) to generate UDP-Araf in yeast.

[0546] Starting with YL-42 (a yeast strain capable of producing QA-C3-GGX-C28-FRX-C9), we then engineered (i) AtUXS and QsC28XylT4 (to produce QA-C3-GGX-C28-FRXX-C9) or AtAXS and QsC28ApiT4 (to produce QA-C3-GGX-C28-FRXA-C9), (ii) QsDMOT4 (to produce QA-C3-GGX-C28-FRXX-C18 or QA-C3-GGX-C28-FRXA-C18), (iii) AtUXE and AtUAM1 (to produce U Genes encoding (i) arabinofuranose transferase (designated "QsArafT" - SEQ ID NO: 229, encoded by the nucleotide sequence shown in SEQ ID NO: 231) (producing QA-C3-GGX-C28-FRXX-C18-A or QA-C3-GGX-C28-FRXA-C18-A) were further integrated into the genome of YL-42 to generate two new yeast strains summarized below, and 2-methylbutyric acid was added to the culture medium. ·AtUXS-QsC28XylT4-AtUXE-AtUAM1-QsDMOT4-QsArafT(YL-45) ·AtUXS-QsApiT4-AtUXE-AtUAM1-QsDMOT4-QsArafT(YL-46)

[0547] result In the single ion LC-MS extraction chromatogram of YL-45, two or more peaks were observed when the exact mass of QS-21-Xyl (i.e., QA-C3-GGX-C28-FRXX-C18-A) was extracted (Figure 49). Similarly, two or more peaks were observed when the exact mass of QA-C3-GGX-C28-FRXX-C18 was extracted, and these masses corresponded to the mass of QA-C3-GGX-C28-FRX-C18-A.

[0548] The peak at retention time 11.1-11.2 min coelutes with a QS-21 standard (the standard is a purified QS-21 fraction from a crude extract of Quillaja bark prepared as described in WO19 / 106192) (Figure 50). The standard was also added to the extracted sample. The isotopic distribution of the extracted mass peak did not change before and after the addition of the standard (Figure 50 inset), confirming that QS-21-Xyl was produced at a titer of 94.6 μg / L in YL-45.

[0549] result In the single ion LC-MS extraction chromatogram of YL-46, multiple similar peaks were also observed when the accurate mass of QS-21-Api (i.e., QA-C3-GGX-C28-FRXA-C18-A) was extracted (Figure 51). Furthermore, a similar peak pattern was observed when the accurate mass of QA-C3-GGX-C28-FRXA-C18 was extracted, which corresponded to the mass of QA-C3-GGX-C28-FRX-C18-A.

[0550] Xylose, arabinofuranose (Araf), and arabinopyranose (Arap) are structural isomers, and therefore have the same exact mass. By adding another pentose instead of Araf, it is possible to obtain a different peak with the same exact mass as QS-21-Api. Based on this, we investigated the substrate range of Araf transferase (QsArafT). We constructed the YL-47 strain by incorporating QsArafT without introducing the genes required for the conversion of UDP-Xyl to UDP-Araf (i.e., without introducing AtUXE and AtUAM1).

[0551] result As a result of this experiment, a new peak corresponding to QA-C3-GGX-C28-FRX-C18-Xyl was observed, suggesting that the addition to the terminus of the acyl chain could also be achieved by incorporating QsArafT, which uses UDP-Xyl as a substrate instead of UDP-Araf (Figure 52).

[0552] To search for an ArafT homologue more specific for UDP-Araf, a BLAST search was performed using the protein sequence of ArafT (SEQ ID NO: 229) against the Quillaja 1kp transcriptome database (https: / / db.cngb.org / onekp / ). OQHZ_scaffold_2012646 was identified as a candidate with 64% protein homology and named "QsArafT2" (SEQ ID NO: 232 encoded by the nucleotide sequence shown in SEQ ID NO: 234). First, this candidate was tested for activity against UDP-Xyl. In a similar manner to above, YL-48 was constructed by incorporating QsArafT2 without introducing the genes required for the conversion of UDP-Xyl to UDP-Araf (i.e., without introducing AtUXE and AtUAM1), and 2-methylbutyrate was added to the culture medium.

[0553] result Although the production of QA-C3-GXX-C18-FRX-C18 was detected (in YL-48), no LC-MS peak corresponding to the addition of Xyl was observed (Figure 53), suggesting that ArafT2 has no activity against UDP-Xyl used as a substrate.

[0554] Based on this result, we generated a new yeast strain (YL-49) that is similar to YL-45 except that the gene encoding QsArafT was replaced with the gene encoding QsArafT2.

[0555] Single ion extraction chromatograms confirmed the formation of QS-21-Xyl (i.e., QA-C3-GXX-C18-FRXX-C18-Araf) and showed a higher ratio of the desired peak over other LC-MS peaks of the same accurate mass (Figure 54).

[0556] 4.6 Incorporation of type I polyketide synthase to produce (S)-2-methylbutyryl-CoA in vivo To eliminate the need for exogenous addition of 2-methylbutyrate, we investigated the biosynthesis of (S)-2-methylbutyryl-CoA (2MB-CoA) in yeast in vivo. First, we investigated the branched-chain α-keto acid dehydrogenase (BCKD) complex using the transaminase from Bacillus subtilis (Bs). The transaminase from Bs can easily convert isoleucine to 2MB-CoA during amino acid metabolism in bacteria. However, 2MB-CoA was not detected in yeast recombinantly expressing BsBKCD (data not shown). Without wishing to be bound by a single theory, this is probably due to the lack of post-translational modification of the E2 subunit of the BKCD complex, which is required for the production of 2MB-CoA.

[0557] Alternatively, we engineered the 7.6 kb type I polyketide synthase (PKS) LovF (also called “megasynthase LovF”) from Aspergillus terreus (Ast) to generate 2MB-CoA in vivo. Native LovF can condense two units of malonyl-CoA into 2MB-ACP (i.e., 2-methylbutyrate covalently linked to the ACP (acyl carrier protein) domain). To obtain free 2-methylbutyrate, we fused the thioesterase (TE) domain of the promiscuous DEBS (6-deoxyerythronolide synthase) from Saccharopolyspora erythraea (Se) to the C-terminus of LovF (also called “LovF-TE”), which cleaves 2-methylbutyrate from the ACP domain. The resulting 2-methylbutyrate can then be converted to 2MB-CoA by QsCCL, resulting in a similar situation to that observed when 2-methylbutyrate is added exogenously. To make LovF functional in a heterologous host, the addition of a phosphopantetheinyl transferase (Ppant) is required. To achieve this, a chromosomal copy of a Ppant candidate from Aspergillus nidulans (referred to as "AnNpgA" - encoded by the nucleotide sequence shown in SEQ ID NO:237 (SEQ ID NO:239)) was integrated into the genome of the parent yeast strain CEN.PK2-1c to generate YL-AnNpgA. A plasmid expressing AstLovF-TE shown in SEQ ID NO:235 (encoded by SEQ ID NO:236) and QsCCL shown in SEQ ID NO:178 (encoded by SEQ ID NO:180) was transfected into YL-AnNpgA to generate YL-PKS. Additionally, AnNpgA and AstLovF-TE were integrated into the genome of YL-42 (a yeast strain that produces QA-C3-GGX-C28-FRX) to generate YL-42-AstLovF-TE.Furthermore, AnNpgA and AstLovF-TE were integrated into the genomes of YL-45 (a yeast strain that produces QA-C3-GGX-C28-FRXX-C18-Araf, i.e., QS-21-Xyl, in the presence of exogenously added 2-methylbutyrate) and YL-46 (a yeast strain that produces QA-C3-GGX-C28-FRXA-C18-Araf, i.e., QS-21-Api, in the presence of exogenously added 2-methylbutyrate), to generate YL-50 and YL-51, respectively.

[0558] result The production of 2MB-CoA by the YL-PKS (containing LovF-TE) was confirmed by LC-MS (Figure 43), indicating that the type I PKS LovF-TE was successfully engineered and catalyzed the release of free 2-methylbutyrate from the ACP domain, which was then ligated to CoA by QsCCL. Although the peak integral of 2MB-CoA was lower than that of 2MB-CoA in the fed-batch experiment of 2-methylbutyrate, the production of QA-C3-GGX-C28-FRX-C9 in YL-42-AstLovF-TE using NgpA and LovF-TE was very close to that in the fed-batch experiment of YL-42, approximately 50% (data not shown). Complete biosynthesis of QS-21-Xyl and QS-21-Api was observed from YL-50 and YL-51, respectively, without exogenous addition of 2-methylbutyrate (shown in Figures 55 and 56, respectively).

[0559] conclusion By using more than 30 heterologous enzymes and proteins derived from various kinds of plants and microorganisms (e.g., Betula oleracea, Quillaja japonica, Arabidopsis thaliana, Saponaria vaccaria, Thermothelomyces thermophilus, Aspergillus nidulans, and Aspergillus terreus), we reconstructed the metabolic pathway for the synthesis of QS-21-Xyl and QS-21-Api (the two major isomeric components present in the QS-21 fraction conventionally purified from Quillaja oleracea bark) in yeast and successfully produced QS-21-Xyl and QS-21-Api in yeast for the first time.

[0560] Example 5 - Methods 5.1 Expression in Nicotiana benthamiana Transient expression experiments in Nicotiana benthamiana were performed as described in WO2020 / 260475.

[0561] 5.2 Yeast recombination Genes were integrated into pESC plasmids containing two multiple cloning sites under the control of the Tet promoter using the tet repressor gene or induced by the galactose-inducible promoters Gal1p and Gal10p, respectively. Nucleotide sequences were codon-optimized for expression in Saccharomyces cerevisiae using IDT online tools. Nucleotide sequences were integrated using an in-house developed CRISPR / Cas9 toolkit. Integration of the nucleotide sequences was confirmed by colony PCR, and yeast strains in which the nucleotide sequences were confirmed to have been integrated were prepared into glycerol stocks and stored at -80°C.

[0562] 5.3 Sugar production and extraction of metabolites The production of sugars and QA derivatives was carried out as follows. First, a glycerol stock of the desired yeast strain was spread on a YPD plate (containing yeast extract, peptone and 2% dextrose) and cultured at 30°C for about 20 hours to obtain a single colony. Next, a single colony was picked from the plate, inoculated into 5 mL of YPD medium and cultured at 30°C with shaking at 200 rpm for 48 hours. The culture was then centrifuged and resuspended in the same volume of YPGal medium (containing yeast extract, peptone and 2% galactose) as above and further cultured at 30°C with shaking at 200 rpm to induce the expression of Gal1 and Gal10 promoters. Samples were taken between 48 and 36 hours after induction of expression to extract metabolites. The yeast cell culture (or cell pellet for sugar production) was extracted with a 2:2:1 ratio of methanol / chloroform / water (2:2:1 (v / v / v)). The aqueous and organic phases were separated by centrifugation and the aqueous phase was collected, which was then evaporated at room temperature using a SpeedVac and resuspended in 0.3% formic acid at pH 9 (pH adjusted with ammonium acetate).

[0563] 5.4 Detection by LC-MS LC-MS analysis was performed using an Agilent HPLC 1260 infinity system equipped with an iQ MSD. Detection: MS (ESI ionization method, spray voltage: (positive) 4.5 kV, (negative) -3.5 kV, mass range: 400-1000, negative ion mode); LC method: Solvent A: [HO + 0.3% formic acid, pH 9 (pH adjusted with ammonium hydroxide)]; Solvent B: [acetonitrile (CHCN) + 0.1% formic acid]; Injection volume: 5 μL: Gradient: 2%-15% [B] from 0-20 min, 15%-50% [B] from 20-26 min, 50%-90% [B] from 26-27 min, 90% [B] from 27-30 min, 90%-2% [B] from 30-31 min, 2% [B] from 31-50 min. The detection method was performed using a porous graphitized carbon column (Hypercarb, 5 μm, 1×150 mm analytical column) at a flow rate of 0.1 mL / min (or as described in WO22 / 136563).

[0564]

Table 4

[0565]

Table 5

[0566]

Table 6

[0567] References Kensil et al. (1991) “Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex”; Journal of immunology, Vol. 146: p431-437 Ragupathi et al. (2011) “Natural and synthetic saponin adjuvant QS-21 for vaccines against cancer”; Expert Review of Vaccines, Vol. 10: p463-470) Garcon et al. “Recent clinical experience with vaccines using MPL and QS-21-containing adjuvant systems”; Expert Review of Vaccines, Vol. 10(4): p71-486 Decker and Kleczkowski (2017) "Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases”; Frontiers in Plant Science, Vol. 8(1610): p1-16 Kirby et al. (2008) Wong et al. (2018) Gosh, 2017 WO 19 / 106192 WO 19 / 122259 WO 20 / 260475 WO 22 / 136563 WO 20 / 263524

Claims

1. A method for producing quillaric acid (QA) in yeast, comprising the steps of: In yeast engineered to produce β-amyrin, (i) cytochrome P450 C16 oxidase, which oxidizes the carbon at the C16 position of β-amyrin to generate a hydroxyl group; (ii) cytochrome P450 C23 oxidase, which oxidizes the carbon at C23 of β-amyrin to generate an aldehyde group; (iii) a cytochrome P450 C28 oxidase that oxidizes the carbon at C28 of β-amyrin to generate a carboxyl group, and / or (iv) cytochrome P450 reductase (CPR) acting as a redox partner overexpressing a heterologous gene encoding The method, wherein the C16 oxidase, the C23 oxidase, the C28 oxidase and the CPR are derived from a plant.

2. 2. The method of claim 1, wherein the C16 oxidase is selected from QsC16 as set forth in SEQ ID NO:20, QsC28C16 as set forth in SEQ ID NO:23, and SvC16 as set forth in SEQ ID NO:26, the C23 oxidase is selected from MtC23 oxidase as set forth in SEQ ID NO:38, QsC23 as set forth in SEQ ID NO:29, SvC23-1 as set forth in SEQ ID NO:32, and SvC23-2 as set forth in SEQ ID NO:35, and the C28 oxidase is selected from MtC28 as set forth in SEQ ID NO:46, QsC28 as set forth in SEQ ID NO:41, and SvC28 as set forth in SEQ ID NO:

44.

3. The method of claim 1 , wherein the yeast further overexpresses (v) a heterologous gene encoding cytochrome b5.

4. The yeast further overexpresses (vi) a heterologous gene encoding a scaffold protein that physically interacts with one or more of the C16 oxidase, the C23 oxidase, the C28 oxidase, and the CPR; The method of claim 1, wherein the scaffold protein is a membrane steroid-binding protein (MSBP) selected from AtMSBP1 as set forth in SEQ ID NO: 63, AtMSBP2 as set forth in SEQ ID NO: 65, QsMSBP1 as set forth in SEQ ID NO: 73, SvMSBP1 as set forth in SEQ ID NO: 67, and SvMSBP2 as set forth in SEQ ID NO:

70.

5. The method according to any one of claims 1 to 4, wherein the yeast produces a β-amyrin synthase (BAS) selected from AaBAS shown in SEQ ID NO: 1, AtBAS shown in SEQ ID NO: 4, GgBAS shown in SEQ ID NO: 7, GvBAS shown in SEQ ID NO: 10, QsBAS shown in SEQ ID NO: 15, and SvBAS shown in SEQ ID NO: 13, and is engineered to overexpress the BAS.

6. The method of claim 4, wherein the C16 oxidase is QsC28C16, the C23 oxidase is QsC23, the C28 oxidase is QsC28, the CPR is AtATR1, the MSBP is SvMSBP1, the cytochrome b5 is Qsb5, and the BAS is GvBAS.

7. 1. A method for producing a C3-glycosylated QA derivative in yeast, comprising the steps of: the C3-glycosylated QA derivative is QA-C3-GlcA, The method comprises the steps of: in a yeast recombinantly engineered to produce QA and UDP-GlcA, (i) UDP-GlcA transferase (GlcAT), which transfers UDP-GlcA and attaches a GlcA residue to the C3 position of QA, forming QA-C3-GlcA. The method comprises overexpressing a heterologous gene encoding the

8. The method of claim 7, wherein the GlcAT is selected from QsCslG1 shown in SEQ ID NO: 78, QsCslG2 shown in SEQ ID NO: 81, and SvCslG shown in SEQ ID NO:

76.

9. the C3-glycosylated QA derivative is QA-C3-GlcA-Gal, and the overexpression (ii) UDP-galactosetransferase (GalT), which transfers UDP-Gal and attaches a Gal residue to QA-C3-GlcA to form QA-C3-GlcA-Gal; The method of claim 7, further comprising overexpressing a heterologous gene encoding

10. The method of claim 9, wherein the GalT is QsGalT as shown in SEQ ID NO: 116 or SvGalT as shown in SEQ ID NO:

98.

11. the C3-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha, the yeast is further engineered to produce UDP-Rha, and the overexpression (iii) UDP-rhamnose transferase (RhaT), which transfers UDP-Rha and attaches the Rha residue to QA-C3-GlcA-Gal, forming QA-C3-GlcA-Gal-Rha. The method of claim 7, further comprising overexpressing a heterologous gene encoding

12. the C3-glycosylated QA derivative is QA-C3-GlcA-Gal-Xyl, the yeast has been further engineered to produce UDP-Xyl, and the overexpression (iii) UDP-xylosetransferase (XylT), which transfers UDP-xylose to attach a Xyl residue to QA-C3-GlcA-Gal to form QA-C3-GlcA-Gal-Xyl; The method according to any one of claims 7 to 11, further comprising overexpressing a heterologous gene encoding

13. The method of claim 12, wherein the XylT is selected from QsC3XylT shown in SEQ ID NO: 122 and SvC3XylT shown in SEQ ID NO:

100.

14. 1. A method for producing UDP-fucose (UDP-Fuc) in yeast, comprising: (i) UDP-glucose-4,6-dehydratase (UG46DH), which converts UDP-Glc to UDP-4-keto-6-deoxyglucose; and / or (ii) 4-ketoreductase, which converts UDP-4-keto-6-deoxyglucose to UDP-D-Fuc The method comprises overexpressing a heterologous gene encoding the

15. 15. The method of claim 14, wherein the UG46DH is SvUG46DH as set forth in SEQ ID NO: 87, and the 4-keto reductase is selected from svNMD as set forth in SEQ ID NO: 90, and QsFucSyn as set forth in SEQ ID NO:

175.

16. 1. A method for producing a C28-glycosylated QA derivative in yeast, comprising the steps of: the C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc or QA-C3-GlcA-Gal-Xyl-C28-Fuc, and the method comprises the steps of: (i) UDP-fucose transferase (FucT), which transfers UDP-Fuc to attach a Fuc residue to the C28 position of QA, forming QA-C3-GlcA-Gal-Rha-C28-Fuc or QA-C3-GlcA-Gal-Xyl-C28-Fuc; The method comprises overexpressing a heterologous gene encoding the

17. The method of claim 16, wherein the FucT is selected from QsFucT as set forth in SEQ ID NO: 93 and SvFucT as set forth in SEQ ID NO:

96.

18. the C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha, and the overexpression (ii) UDP-rhamnose transferase (RhaT), which transfers UDP-Rha to link Rha residues to QA-C3-GlcA-Gal-Rha-C28-Fuc or QA-C3-GlcA-Gal-Xyl-C28-Fuc, forming QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha.

17. The method of claim 16, further comprising overexpressing a heterologous gene encoding

19. The method of claim 18, wherein the RhaT is QsRhaT as shown in SEQ ID NO:

119.

20. the C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl, and the overexpression (iii) UDP-xylosetransferase (XylT), which transfers UDP-Xyl and attaches a Xyl residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha to form GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl, respectively.

20. The method of claim 18, further comprising overexpressing a heterologous gene encoding

21. The method of claim 20, wherein the XylT is QsC28XylT3 as shown in SEQ ID NO:

125.

22. the C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, and the overexpression is (iv) UDP-xylosetransferase (XylT), which transfers UDP-Xyl and attaches a Xyl residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, respectively.

21. The method of claim 20, further comprising overexpressing a heterologous gene encoding

23. The method of claim 22, wherein the XylT is QsC28XylT4 as shown in SEQ ID NO:

128.

24. the C28-glycosylated QA derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api, and the overexpression is (iv) UDP-apiose synthase (AXS), which converts UDP-GlcA to UDP-Api, and / or (v) UDP-apiose transferase (ApiT), which transfers UDP-apiose and attaches the apiose residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api, respectively.

23. The method of claim 22, further comprising overexpressing a heterologous gene encoding

25. The method of claim 24, wherein the AXS is QsAXS as set forth in SEQ ID NO: 113, and the ApiT is QsC28ApiT4 as set forth in SEQ ID NO:

151.

26. 1. A method for producing (S)-2-methylbutyryl-CoA (2MB-CoA) in yeast, comprising: In the presence of exogenously added 2-methylbutyric acid, Carboxyl coenzyme A (CoA) ligase (CCL) converts 2-methylbutyrate to 2MB-CoA The method comprises overexpressing a heterologous gene encoding the

27. The method of claim 26, wherein the CCL is QsCCL from Q. saponaria as set forth in SEQ ID NO:

178.

28. The overexpression can be achieved without exogenous addition of 2-methylbutyric acid. (i) phosphopantetheinyl transferase (Ppant), and / or (ii) Megasynthetase LovF-TE, which contains an acyl carrier protein (ACP) domain and further comprising overexpressing a heterologous gene encoding The method of claim 26, wherein the megasynthetase LovF-TE condenses two units of malonyl-CoA with 2MB-ACP, cleaves 2-methylbutyrate from the ACP domain, and converts 2-methylbutyrate to 2MB-CoA by the CCL.

29. 29. The method of claim 28, wherein the Ppant is AnNpgA as set forth in SEQ ID NO: 237 and the megasynthetase LovF-TE is AstLovF-TE as set forth in SEQ ID NO:

235.

30. A method for producing UDP-arabinofuranose (UDP-Araf) in yeast, comprising: In yeast engineered to produce UDP-Xyl, (i) a UDP-Xyl epimerase (UXE) that converts UDP-Xyl to UDP-arabinopyranose (UDP-Arap); and / or (ii) UDP-arabinose mutase (UAM), which converts UDP-Arap to UDP-arabinofuranose (UDP-Araf); The method comprises overexpressing a heterologous gene encoding the

31. The method of claim 30, wherein the UXE is selected from AtUXE as set forth in SEQ ID NO: 199, AtUXE2 as set forth in SEQ ID NO: 202, HvUXE-1 as set forth in SEQ ID NO: 240, HvUXE-2 as set forth in SEQ ID NO: 242, and AtUGE3 as set forth in SEQ ID NO: 205, and the UAM is selected from AtUAM1 as set forth in SEQ ID NO: 208, and HvUAM as set forth in SEQ ID NO:

211.

32. A method for producing UDP-Araf in yeast, comprising: In the presence of exogenously added arabinose, (i) arabinokinase (AraK), and / or (ii) UDP-sugar pyrophosphorylase (USP) The method comprises overexpressing a heterologous gene encoding the

33. The method of claim 32, wherein the AraK is selected from AtAraK as set forth in SEQ ID NO: 214 and LeiAraK as set forth in SEQ ID NO: 217, and the USP is selected from AtUSP as set forth in SEQ ID NO: 223 and LeiUSP as set forth in SEQ ID NO:

226.

34. 34. The method of claim 33, wherein said overexpressing further comprises overexpressing a heterologous gene encoding an arabinose transporter (AraT).

35. The method of claim 35, wherein the AraT is encoded by the nucleotide sequence of SEQ ID NO:

221.

36. 1. A method for producing an acylated glycosylated QA derivative in yeast, comprising the steps of: the acylated glycosylated QA derivative is QA-C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9, The method further comprising: In yeast engineered to produce QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXA or QA-C3-GGX-C28-FRXA, in the presence of exogenously added 2-methylbutyrate, (i) carboxyl coenzyme A ligase (CCL), which converts 2-methylbutyrate to (S)-2-methylbutyryl CoA (2MB-CoA); (ii) a chalcone synthase-like type III polyketide synthase (PKS) that condenses malonyl-CoA with 2MB-CoA to form C9-keto-CoA; (iii) a ketoreductase (KR) that converts C9-keto-CoA to C9-CoA; and / or (iv) an acyltransferase which transfers the first C9-CoA unit to form QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXA or QA-C3-GGX-C28-FRXA-C9 by coupling to QA-C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9 overexpressing a heterologous gene encoding A method comprising:

37. The method of claim 36, wherein the CCL is QsCCL as set forth in SEQ ID NO: 178, the chalcone synthase-like type III PKS is QsChSD as set forth in SEQ ID NO: 181, QsChSE as set forth in SEQ ID NO: 184, or both QsChSD as set forth in SEQ ID NO: 181 and QsChSE as set forth in SEQ ID NO: 184, the ketoreductase is QsKR11 as set forth in SEQ ID NO: 187, QsKR23 as set forth in SEQ ID NO: 190, or both QsKR11 as set forth in SEQ ID NO: 187 and QsKR23 as set forth in SEQ ID NO: 190, and the acyltransferase is QsDMOT9 as set forth in SEQ ID NO:

193.

38. the acylated glycosylated QA derivative is QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXA-C18 or QA-C3-GGX-C28-FRXA-C18, The overexpression (v) attaching a second C9-CoA unit to C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9 to form C3-GGR- acyltransferase QsDMOT4 according to SEQ ID NO: 196 which forms C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXA-C18 or QA-C3-GGX-C28-FRXA-C18 37. The method of claim 36, further comprising overexpressing a heterologous gene encoding

39. The method of claim 38, wherein the QsDMOT4 is encoded by the nucleotide sequence of SEQ ID NO:

197.

40. the acylated glycosylated QA derivative is QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf, the yeast is further engineered to produce UDP-Araf; The overexpression (vi) transferring UDP-Araf to attach Araf residues to QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXA-C18 or QA-C3-GGX-C28-FRXA-C18; Arabinotransferase (ArafT) forming GR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf 39. The method of claim 38, further comprising overexpressing a heterologous gene encoding

41. 41. The method of claim 40, wherein the ArafT is selected from QsArafT as set forth in SEQ ID NO: 229 and QsArafT2 as set forth in SEQ ID NO:

232.

42. The method of claim 41, wherein the ArafT is QsArafT2 as set forth in SEQ ID NO:

232.

43. 1. A method for producing QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GGR-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXA-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl in a yeast, comprising: in a yeast engineered to produce QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GRX-C28-FRXX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRX-C18, By transferring UDP-Xyl to QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GRX-C28-FRXX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRX-C18, a Xyl residue was attached to QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, or QA-C3-GGR-C28-FRX-C18. overexpressing a heterologous gene encoding an arabinotransferase (ArafT) that forms 8-Xyl, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GRX-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXA-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl A method comprising:

44. The method of claim 43, wherein the ArafT is QsArafT as set forth in SEQ ID NO:

229.

45. The overexpression can be achieved without exogenous addition of 2-methylbutyric acid. (i) phosphopantetheinyl transferase (Ppant), and / or (ii) Megasynthetase LovF-TE, which contains an acyl carrier protein (ACP) domain and further comprising overexpressing a heterologous gene encoding The method of any one of claims 37 to 45, wherein the megasynthetase LovF-TE condenses two units of malonyl-CoA with 2MB-ACP, cleaves 2-methylbutyrate from the ACP domain, and converts 2-methylbutyrate to 2MB-CoA by CoA ligase (CCL).

46. 46. ​​The method of claim 45, wherein the Ppant is AnNpgA as set forth in SEQ ID NO: 237 and the megasynthetase LovF-TE is AstLovF-TE as set forth in SEQ ID NO:

235.

47. 1. A method for producing QA-C3-GGX-C28-FRXX-C18-Araf (QS-21-Xyl) in yeast, comprising: GvBAS shown in SEQ ID NO: 10, QsC28C16 shown in SEQ ID NO: 23, QsC23 shown in SEQ ID NO: 29, QsC28 shown in SEQ ID NO: 41, AtATR1 shown in SEQ ID NO: 49, Qsb5 shown in SEQ ID NO: 55, SvMSBP1 shown in SEQ ID NO: 67, AtUGD shown in SEQ ID NO: 108 A101L , QsCslG2 as shown in SEQ ID NO: 78, QsGalT as shown in SEQ ID NO: 116, AtUXS as shown in SEQ ID NO: 105, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFuct as shown in SEQ ID NO: 93, AtRHM2 as shown in SEQ ID NO: 102, QsRhaT as shown in SEQ ID NO: 119, QsC28XylT3 as shown in SEQ ID NO: 125, QsC28XylT4 as shown in SEQ ID NO: 128, QsChSD as shown in SEQ ID NO: 181, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFuct as shown in SEQ ID NO: 93, AtRHM2 as shown in SEQ ID NO: 102, QsRhaT as shown in SEQ ID NO: 119, QsC28XylT3 as shown in SEQ ID NO: 125, QsC28XylT4 as shown in SEQ ID NO: 128, QsChSD as shown in SEQ ID NO: 18 4, QsKR11 as shown in SEQ ID NO: 187, QsKR23 as shown in SEQ ID NO: 190, QsDMOT9 as shown in SEQ ID NO: 193, QsDMOT4 as shown in SEQ ID NO: 196, AtUXE as shown in SEQ ID NO: 199, AtUAM1 as shown in SEQ ID NO: 208, QsArafT2 as shown in SEQ ID NO: 232, AnNpgA as shown in SEQ ID NO: 237, QsCCL as shown in SEQ ID NO: 178 and / or AstLovF-TE as shown in SEQ ID NO:

235.

48. A method for producing QA-C3-GGX-C28-FRXA-C18-Araf (QS-21-Api) in yeast, comprising: GvBAS shown in SEQ ID NO: 10, QsC28C16 shown in SEQ ID NO: 23, QsC23 shown in SEQ ID NO: 29, QsC28 shown in SEQ ID NO: 41, AtATR1 shown in SEQ ID NO: 49, Qsb5 shown in SEQ ID NO: 55, SvMSBP1 shown in SEQ ID NO: 67, AtUGD shown in SEQ ID NO: 108 A101L , QsCslG2 as shown in SEQ ID NO: 81, QsGalT as shown in SEQ ID NO: 116, AtUXS as shown in SEQ ID NO: 105, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFucT as shown in SEQ ID NO: 93, AtRHM2 as shown in SEQ ID NO: 102, QsRhaT as shown in SEQ ID NO: 119, QsC28XylT3 as shown in SEQ ID NO: 125, QsC28ApiT4 as shown in SEQ ID NO: 151, QsChSD as shown in SEQ ID NO: 181, QsC3XylT as shown in SEQ ID NO: 122, SvNMD as shown in SEQ ID NO: 90, SvUG46DH as shown in SEQ ID NO: 87, QsFucT as shown in SEQ ID NO: 93, 4, QsKR11 as shown in SEQ ID NO: 187, QsKR23 as shown in SEQ ID NO: 190, QsDMOT9 as shown in SEQ ID NO: 193, QsDMOT4 as shown in SEQ ID NO: 196, AtUXE as shown in SEQ ID NO: 199, AtUAM1 as shown in SEQ ID NO: 208, QsArafT2 as shown in SEQ ID NO: 232, AnNpgA as shown in SEQ ID NO: 237, QsCCL as shown in SEQ ID NO: 178 and / or AstLovF-TE as shown in SEQ ID NO:

235.

49. A C3-glycosylated QA derivative obtainable by the method according to any one of claims 7 to 13.

50. A C28-glycosylated QA derivative obtainable by the method according to any one of claims 16 to 25.

51. An acylated glycosylated QA derivative obtainable by the method according to any one of claims 36 to 46.

52. Use of the C3-glycosylated QA derivative according to claim 49, the C28-glycosylated QA derivative according to claim 50 and the acylated glycosylated QA derivative according to claim 51 as adjuvants.

53. An isolated polypeptide, the polypeptide being selected from the group consisting of β-amyrin synthase (SvBAS) shown in SEQ ID NO: 13, β-amyrin synthase (QsBAS) shown in SEQ ID NO: 15, CYP C16 oxidase (QsC28C16) shown in SEQ ID NO: 23, CYP C16 oxidase (SvC16) shown in SEQ ID NO: 26, CYP C23 oxidase (SvC23-1) shown in SEQ ID NO: 32, CYP C23 oxidase (SvC23-2) shown in SEQ ID NO: 35, CYP C23 oxidase (SvC23-3) shown in SEQ ID NO: 44, and CYP C23 oxidase (SvC23-4) shown in SEQ ID NO:

46. C28 oxidase (SvC28), cytochrome b5 protein (Qsb5) shown in SEQ ID NO: 55, cytochrome b5 protein (Svb5) shown in SEQ ID NO: 61, UDP-GlcA transferase (SvCslG) shown in SEQ ID NO: 76, MSBP protein (SvMSBP1) shown in SEQ ID NO: 67, MSBP protein (SvMSBP2) shown in SEQ ID NO: 70, MSBP protein (QsMSBP1) shown in SEQ ID NO: 73, UDP-glucose-4,6-dehydratase (SvUG46D) shown in SEQ ID NO: 87 H), UDP-4-keto-6-deoxyglucose reductase (SvNMD) as set forth in SEQ ID NO: 90, UDP-galactose transferase (SvGalT) as set forth in SEQ ID NO: 98, UDP-fucose transferase (SvFucT) as set forth in SEQ ID NO: 96, UDP-xylose transferase (SvC3XylT) as set forth in SEQ ID NO: 100, UDP-arabinofuranose transferase (QsArafT2) as set forth in SEQ ID NO: 229, UDP-glucose dehydrogenase (AtUGD) as set forth in SEQ ID NO: 108, A101L ), UDP-xylose transferase (QsC28XylT4-3aa) shown in SEQ ID NO: 131, UDP-xylose transferase (QsC28XylT4-6aa) shown in SEQ ID NO: 134, UDP-xylose transferase (QsC28XylT4-9aa) shown in SEQ ID NO: 137, UDP-xylose transferase (QsC28XylT4-12aa) shown in SEQ ID NO: 140, UDP-xylose transferase (SU A polypeptide selected from the group consisting of a UDP-xylose transferase (MBP-QsC28XylT4) set forth in SEQ ID NO: 147, a UDP-xylose transferase (QsC28XylT3-3xGGGS-QsC28XylT4) set forth in SEQ ID NO: 149, and a type I polyketide synthase (AstLovF-TE) set forth in SEQ ID NO: 235.

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