FIELD OF THE INVENTION

The current invention is situated in the fields of plant secondary metabolites with pharmacological or other industrial properties and metabolic engineering of these phytochemicals. More specifically, the invention relates to a method for enhancing the biosynthesis and/or secretion of sapogenin intermediates in the culture medium of plant and microbial cell cultures. Further, the present invention also relates to the identification of novel genes involved in the biosynthesis of sapogenin intermediates, as well as to novel sapogenin compounds.

BACKGROUND Plants synthesize an overwhelming variety of triterpene saponins with an enormous range of biological activities relevant for the pharmaceutical and chemical industry (e.g. additives to foods and cosmetics). Interest in triterpenoid saponins and its precursors has increased recently because of data showing their diverse biological activities and beneficial properties, which include antifungal, antibacterial, antiviral, antitumor, molluscicidal, insecticidal, and antifeedant activities (Suzuki et al. 2002; Sparg et al. 2004; Huhman et al. 2005). Saponins are synthesized by multiple glycosylations of sapogenin building blocks, which in turn are produced by multiple modifications (e.g. hydroxylations) of basic sapogenin backbones such as β-amyrin, lupeol, and dammarenediol. These diverse backbones are generated by specific cyclizations of 2,3-oxidosqualene, which is also an intermediate in the synthesis of membrane sterols. As an illustration, more than seventy saponins have been identified in the model legume, M. truncatula (Huhman and Sumner 2002; Pollier et al. 2011), the core of this diversity being centralized in a few aglycones (sapogenins). Also these precursor sapogenins are very valuable compounds and are important starter molecules for further synthetic modifications. For example, the naturally occurring triterpenoid sapogenin oleanolic acid and its derivatives possess several promising pharmacological activities, such as hepato-protective effects, and anti-inflammatory, antioxidant, or anticancer activities (Pollier and Goossens 2012).

The first committed step in triterpenoid saponin biosynthesis is the cyclization of 2,3-oxidosqualene (Fig. 1). This reaction is catalyzed by specific oxidosqualene cyclases (OSCs), including β-amyrin synthase (bAS; EC 5.4.99.-), and has been functionally characterized in several plants (Kushiro et al. 1998, Herrera et al. 1998, Iturbe-Ormaetxe et al. 2003, Morita et al. 2000, Suzuki et al. 2002). Then, the action of oxidative enzymes (typically cytochrome P450 monooxygenases or CYPs) and glycosyltransferases convert β-amyrin to various triterpene saponins in different plant species. For example, subsequent modifications that impart functional properties and diversify the basic triterpenoid backbone include the addition of small functional groups, including hydroxyl, keto, aldehyde, and carboxyl moieties, generally followed by glycosylation reactions (Augustin et al. 2011). To date, a number of CYPs that use β-amyrin as a substrate have been identified in dicotyledonous plants, whereas just one (CYP51H10) has been identified in monocots.

Present availability of saponins and sapogenins depends on their extractability from plants and is often uneconomical and inefficient. Often laborious extraction schemes have to be developed for each specific metabolite of interest and a steady supply of sufficient amounts of specific sapo(ge)nins from plants that accumulate mixtures of structurally related compounds is not feasible. Synthetic chemistry mainly attempts to address these issues by chemically linking desired side chains to extracted sapogenins, as was done for oleanolic acid. However, the structural complexity of the sapogenins hampers chemical synthesis and the availability of corresponding sapogenins forms a major bottleneck.

The culture of plant cells has been explored since the 1960's as a viable alternative for the production of complex phytochemicals of industrial interest. For example, the use of large-scale plant cell cultures in bioreactors for the production of alkaloids has been extensively studied (Verpoorte et al. 1999). Despite the promising features and developments, the production of plant-derived pharmaceuticals by plant cell cultures has not been fully commercially exploited. The main reasons for this reluctance shown by industry to produce phytochemicals by means of cell cultures, compared to the conventional extraction of whole plant material, are economical ones based on the slow growth and the low production levels of phytochemicals by such plant cell cultures. Important causes are the toxicity of such compounds to the plant cell, and the role of catabolism of these compounds. Another important problem is that many phytochemicals, such as the triterpene saponins and its precursors, are mostly retained intracellular^ complicating the downstream processing and purification. Another important problem is that for many phytochemicals the precursors or intermediates in the pathway do not accumulate or only in trace amounts, because they are readily converted by the downstream enzymes.

Biotechnological production of either complete saponins, or of sapogenin pathway intermediates that are not readily accessible, may circumvent the limitation of natural sapo(ge)nin availability. However, circumvention of laborious and uneconomical extraction procedures for industrial production from plants is also hampered by lack of knowledge and availability of genes in saponin biosynthesis. As a consequence, although triterpene synthases have been expressed in microbial hosts such as Saccharomyces cerevisiae, there has been little effort made so far to engineer the metabolism of a microbial host for enhanced production of triterpenes. By contrast, there have been many considerable efforts to engineer microbes for higher production of mono-, sesqui- and diterpenes. Notably, triterpene production may not be as amenable to engineering efforts as the volatile sesquiterpenes and monoterpenes that readily diffuse out of the cell.

Therefore, a need exists for the cost-effective biotechnological production of high value sapo(ge)nins or other triterpene building blocks in a convenient host cell.

SUMMARY OF THE INVENTION

Evidence is available that sapogenins, when produced in their natural hosts (plants), are often only found in trace amounts intracellular^ in plant cells, as also demonstrated in Example 1. Moreover, and as shown in Example 3 and 4, although sapogenins can be heterologously produced in genetically engineered yeast cells, they are only detected when extracted from the cells, and they are not found in the growth medium. In order to overcome these problems, the inventors have found that by incubating a eukaryotic cell culture that is capable of intracellular^ synthesizing sapogenins in a culture medium with cyclodextrins, significant amounts of sapogenins can be extracted from the culture medium (Example 5, 6 and 7). Cyclodextrins, which are cyclic oligosaccharides consisting of five or more a-D-glucopyranose residues linked by a(l->4) glucosidic bonds, are known to act as sequestering agents of phytosterols from membranes (Raffaele et al. 2009) and have been used as elicitors to increase the production and extraction of phytosterols from cultures of plant cells (EP2351846; Sabater-Jara et al. 2010). In the present invention, it is shown that intracellular^ synthesized sapogenins can be released in the growth medium of eukaryotic cell cultures in the presence of cyclodextrins and can accumulate in significant amounts. In addition, it was shown that substantially higher amounts can be obtained by repeatedly adding cyclodextrins to the culture medium.

Thus, in a first aspect, the invention relates to a method for producing triterpenoid sapogenins in the extracellular medium of a eukaryotic cell culture comprising: a. Providing eukaryotic cells capable of synthesizing triterpenoid sapogenins under suitable conditions; and

b. Incubating the cells in culture medium comprising cyclodextrins; and

c. Optionally, extracting the sapogenins from the culture medium.

According to a preferred embodiment, the eukaryotic cells naturally produce triterpenoid sapogenins, such as plants cells. Alternatively, the eukaryotic cells are genetically engineered to produce triterpenoid sapogenins. Such genetically engineered eukaryotic cells can be microbial cells, such as yeast cells, or plant cells, or algal cells.

According to further preferred embodiments, the cyclodextrins are selected from the group comprising randomly methylated cyclodextrins or hydroxypropylated cyclodextrins. Preferably, the cyclodextrin is a β-cyclodextrin. According to particular embodiments, the cyclodextrins may be added to the culture medium once or at different consecutive time points.

In other aspects, the invention also envisages eukaryotic cell genetically engineered to synthesize sapogenins.

Further, a sapogenin obtained by any of the above described methods is also encompassed.

According to yet another aspect, the invention relates to an isolated polypeptide selected from the group consisting of:

(a) a polypeptide encoded by a polynucleotide comprising SEQ ID NO: 1 or 2;

(b) a polypeptide comprising a polypeptide sequence having a least 75% identity to the polypeptide encoded by a polynucleotide sequence having SEQ ID NO: 1 or 2;

(c) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3 or 4;

(d) a polypeptide comprising an amino acid sequence with at least 75% identity to SEQ ID NO: 3 or 4;

(e) fragments and/or variants of the polypeptides according to (a), (b), (c), (d).

In one embodiment, the invention relates to any of the above described polypeptides wherein said polypeptide sequence is consisting of an amino acid sequence as set forth in SEQ ID NO: 3 or 4 and polypeptide sequences having at least 75% identity to SEQ ID NO: 3 or 4.

According to yet another aspect, the invention relates to an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising a polynucleotide sequence having the sequence SEQ ID NO: 1 or 2;

(b) a polynucleotide comprising a polynucleotide sequence having at least 70% identity to the sequence having SEQ ID NO: 1 or 2;

(c) a polynucleotide which encodes the polypeptide sequence as set forth in SEQ ID NO: 3 or 4;

(d) a polynucleotide which encodes the polypeptide sequence as set forth in SEQ ID NO: 3 or 4;

(e) fragments and variants of the polynucleotides according to (a), (b), (c) or (d). In a specific embodiment, the invention relates to a chimeric gene comprising the following operably- linked sequences: a) a promoter region capable of directing expression in a eukaryotic cell (as defined herein before); b) a DNA region encoding a polypeptide as defined above; c) a 3' polyadenylation and transcript termination region.

A vector comprising a polynucleotide sequence or a chimeric gene as defined above also forms part of the present invention, as well as a host cell comprising a polynucleotide sequence or a chimeric gene or a vector as defined above.

According to a particular aspect, the invention provides a transgenic plant or a cell derived thereof that is transformed with the above described vector.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Plant bioactive triterpene saponins. Schematic summary of biosynthetic pathways and variability in structure. Single and multiple arrows indicate single and multiple catalytic (enzymatic or semi-synthetic) conversions, respectively. AT, acyltransferase; BAS, β-amyrin synthase; CAS, cycloartenol synthase; CytP450, Cytochrome P450; FPP, farnesylpyrophosphate; GT, glycosyltransferase; LUP, lupeol synthase; MT, methyltransferase; PNA, dammarenediol-ll synthase; SQE, squalene epoxidase; SQS, squalene synthase.

Figure 2: GC chromatogram of extraction from M. truncatula hairy roots showing the presence of abundant sterols and sterol intermediates, but only trace amounts of triterpenoid sapogenins.

Figure 3: GC chromatogram of; A) extraction from cells of strain TM3 showing β-amyrin at 27.2 min, B) extraction from spent medium of strain TM3, C) extraction from cells of strain TM5, D) β-amyrin standard.

Figure 4: GC chromatogram of; A) extraction from cells of strain TM6 showing lupeol at 28.9 min, B) extraction from spent medium of strain TM6, C) extraction from cells of strain TM5, D) lupeol standard.

Figure 5: Quantification of; A) β-amyrin from the cells and spent medium of strain 7M3,where 100 % corresponds to 36.2 mg/L of β-amyrin, B) lupeol from the cells and spent medium of strain TM6, where 100 % corresponds to 46.3 mg/L of lupeol. Higher concentrations of β-amyin and lupeol were quantified from the extracts of spent medium compared to cell pellet upon cyclodextrin treatment. Figure 6: Dose dependent secretion of β-amyrin quantified for strain TM3, where cyclodextrin was added on; Day 1 (II), Day 1 and 2 (12), Day 1, 2 and 3 (13), Day 2 (Rl), Day 2 and 3 (R2), Day 3 (AR) and untreated control (C).

Figure 7: GC chromatograms of extraction performed on spent medium of; A) strain TM10 expressing CYP716A12, B) strain TM11 expressing CYP88D6, C) strain TM12 expressing CYP93E2, D) control strain TM26 expressing no CYP.

Figure 8: GC chromatograms of extraction performed on spent medium of; A) strain TM22 expressing CYP716A12, B) control strain TM28 expressing no CYP.

Figure 9: GC chromatograms of extraction performed on spent medium of M. truncatula hairy roots treated for 48 h with; A) 25 mM cyclodextrin, B) 25 mM cyclodextrin and 100 μΜ methyl jasmonate, C) 100 μΜ methyl jasmonate, and D) untreated control.

Figure 10: Chemical structure of oleanane-type sapogenin backbone. Asterisk (*) indicate the carbon positions for which a CytP450 has already been characterized.

Figure 11. Transcript profiling of jasmonate elicited B. falcatum roots. Subcluster of the B. falcatum transcriptome, comprising tags corresponding to genes reported to be involved in triterpene biosynthesis, or with high sequence similarity to such genes. Treatments and time points (in h) are indicated on top. Blue and yellow boxes reflect transcriptional activation and repression relative to the average expression level, respectively. Gray boxes correspond to missing time points. The arrowhead indicates the CytP450 functionally defined in this study. Figure 12. GC chromatograms corresponding to, A) Extraction from cells of strain TM7. B) Extraction from cells of strain TM10. Enclosed box figure shows the mass spectra extracted from the indicated (*) peak. C) Extraction from cells of control strain TM26. D) Mass spectra extracted from the peak indicated (*) at 31.8 min of strain TM7. E) Mass spectra of an erythrodiol standard.

Figure 13. A) Effect of CPR:CytP450 ratio on the in vivo activity of CYP716A021 in strains TM8, TM9 and TM7. B) Relative amounts of hydroxylated β-amyrin quantified from the cells and spent medium of strain TM9. C) Relative amounts of hydroxylated β-amyrin quantified from spent medium of strain TM9 treated with different variants of CD.

Figure 14. Chemical structure of maesasapogenins. Figure 15. Transcript profiling of jasmonate elicited M. lanceolate/ plants. Subcluster of the M. lanceolata transcriptome, comprising all tags corresponding to genes reported to be involved in terpene biosynthesis, or with high sequence similarity to such genes, and all gene tags corresponding to CytP450s. Treatments and time points (in h) are indicated on top. Blue and yellow boxes reflect transcriptional activation and repression relative to the average expression level, respectively. Gray boxes correspond to missing time points.

Figure 16. GC chromatograms corresponding to, (a) Extraction from spent medium of strain TM21. Arrow heads indicate the positions that could be hydroxylated by ML593 and are common with predicted positions of CYP716A021. (b) Extraction from spent medium of strain TM9. (c) Extraction from spent medium of control strain TM27. Right panel shows mass spectra extracted from the indicated (*) peaks.

Figure 17. GC chromatograms corresponding to, A) Extraction from spent medium of strain TM30. B) Extraction from spent medium of strain TM9. C) Extraction from spent medium of strain TM17. D) An echinocystic acid standard. E) Mass spectra extracted from the peak indicated (*) at 40.5 min of strain TM30. Parts of the structure in blue depict the possible unknown hydroxylation position. F) Mass spectra extracted from echinocystic acid standard. G) Oxidation of β-amyrin by CYP716A021 and CYP716A12.

Figure 18. GC chromatograms corresponding to, (a) Extraction from spent medium of strain TM31. (b) Extraction from spent medium of strain TM17. (c) Extraction from spent medium of strain TM21. (d) Extraction from spent medium of strain TM30. (e) An echinocystic acid standard. Right panel shows the mass spectra of indicated (*) peaks. Parts of structure highlighted in blue indicate probable hydroxylation positions.

Figure 19. GC chromatograms corresponding to, (a) Extraction from spent medium of strain TM32. (b) Extraction from spent medium of strain TM21. (c) Extraction from spent medium of strain TM18. (d) Extraction from spent medium of control strain TM27. (e) Mass spectra extracted from indicated (*) peaks of strain TM18 and TM32. Right panels show the mass spectra extracted from the indicated (*) peaks, (f) Oxidation of β-amyrin by ML593 and CYP88D6.

Figure 20. GC chromatograms corresponding to, (a) Extraction from spent medium of strain TM33. (b) Extraction from spent medium of control strain TM5. (c) A β-amyrin standard, (d) A a-amyrin standard. Right panels show the mass spectra extracted from the indicated (*) peaks, (e) Cyclization of 2,3- oxidosqualene by a-amyrin synthase (aAS), β-amyrin synthase (MS), and dammarenediol synthase (DDS) to a-amyrin , β-amyrin and dammarenediol, respectively.

Figure 21. GC chromatograms corresponding to, (a) Extraction from spent medium of strain TM37. Right panels show the mass spectra extracted from the indicated (*) peaks, (b) Extraction from spent medium of control strain TM38. Parts of structure in green indicate the putative hydroxylation position.

DETAI LED DESCRI PTION OF TH E INVENTION

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of molecular and cellular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Leach, Molecular Modelling: Principles and Applications, 2d ed., Prentice Hall, New Jersey (2001). According to a first aspect, the present invention relates to a method for producing triterpenoid sapogenins in the extracellular medium of a eukaryotic cell culture comprising: a. Providing eukaryotic cells capable of synthesizing triterpenoid sapogenins under suitable conditions; and

b. Incubating the cells in culture medium comprising cyclodextrins; and

c. Optionally, extracting the sapogenins from the culture medium.

Saponins are a group of natural bio-active compounds that consist of an isoprenoidal-derived aglycone, designated "genin" or "sapogenin", covalently linked to one or more sugar moieties. This combination of polar and non-polar structural elements in their molecules explains their soap-like behavior in aqueous solutions. Most known saponins are plant-derived secondary metabolites, though several saponins are also found in marine animals such as sea cucumbers and starfish. In plants, saponins are generally considered to be part of defense systems due to anti-microbial, fungicidal, allelopathic, insecticidal and moluscicidal, etc. activities. Typically, saponins reside inside the vacuoles of plant cells. Extensive reviews on molecular activities, biosynthesis, evolution, classification, and occurrence of saponins are given by e.g. Augustin et al. (2011) and Vincken et al. (2007). Thus, the term "sapogenin", as used herein, refers to an aglycone, or non-saccharide, moiety of the family of natural products known as saponins.

The commonly used nomenclature for saponins distinguishes between triterpenoid saponins (also: triterpene saponins) and steroidal saponins, which is based on the structure and biochemical background of their aglycones. Both sapogenin types are thought to derive from 2,3-oxidosqualene, a central metabolite in sterol biosynthesis. In phytosterol anabolism, 2,3-oxidosqualene is mainly cyclized into cycloartenol. "Triterpenoid sapogenins" branch off the phytosterol pathway by alternative cyclization of 2,3-oxidosqualene, while "steroidal sapogenins" are thought to derive from intermediates in the phytosterol pathway downstream of cycloartenol formation (see also Fig. 1). A more detailed classification of saponins based on sapogenin structure with 11 main classes and 16 subclasses has been proposed by Vincken et al. (2007; particularly from page 276 to page 283, and also Fig. 1 and Fig. 2), which is all incorporated herein by reference. In particular, saponins may be selected from the group comprising dammarane type saponins, tirucallane type saponins, lupane type saponins, oleanane type saponins, taraxasterane type saponins, ursane type saponins, hopane type saponins, cucurbitane type saponins, cycloartane type saponins, lanostane type saponins, steroid type saponins. The aglycon backbones, the sapogenins, can be similarly classified and may be selected from the group comprising dammarane type sapogenins, tirucallane type sapogenins, lupane type sapogenins, oleanane type sapogenins, taraxasterane type sapogenins, ursane type sapogenins, hopane type sapogenins, cucurbitane type sapogenins, cycloartane type sapogenins, lanostane type sapogenins, steroid type sapogenins. Examples of sapogenins produced in plants are given in Table 1.

According to the above definitions, and as used herein, the "triterpenoid sapogenins" may be selected from the group comprising dammarane type sapogenins, tirucallane type sapogenins, lupane type sapogenins, oleanane type sapogenins, ursane type sapogenins, hopane type sapogenins. Thus, according to specific embodiments, the triterpenoid sapogenins as produced by the method of the invention are dammarane type sapogenins, or tirucallane type sapogenins, or lupane type sapogenins, or oleanane type sapogenins, or ursane type sapogenins, or hopane type sapogenins. Triterpenoid sapogenins typically have a tetracyclic or pentacyclic skeleton. As described in the Background section, the sapogenin building blocks themselves may have multiple modifications, e.g. small functional groups, including hydroxyl, keto, aldehyde, and carboxyl moieties, of precursor sapogenin backbones such as β-amyrin, lupeol, and dammarenediol.

The terms "triterpene" and "triterpenoid" are used interchangeably herein. It is to be understood that the triterpenoid sapogenins, as used herein, also encompass new-to-nature triterpenoid compounds which are structurally related to the naturally occurring triterpenoid sapogenins. These new-to-nature triterpenoid sapogenins may be currently unextractable compounds by making use of existing extraction procedures or may be novel compounds that can be obtained after genetic engineering of the synthesizing eukaryotic host cell (see further herein). For the sake of clarity, the term "triterpenoid sapogenins", as used herein, is not meant to cover phytosterols or phytosterol pathway intermediates. Phytosterols, which encompass plant sterols and stanols, are triterpenes that are important structural components of plant membranes and free phytosterols serve to stabilize phospholipid bilayers in plant cell membranes just as cholesterol does in animal cell membranes. Stanols are a fully-saturated subgroup of phytosterols (contain no double bonds). Non-limiting examples of phytosterols which form important structural components of plant membranes are stigmasterol, β-sitosterol, fucosterol, campesterol. Table 1. Triterpenoid sapogenins comprising the core of commonly accumulating saponins produced by plants.

Plant genus Sapogenins comprised in commonly Chemical formula Reference

accumulating saponins of sapogenin

Medicago 2,3-dihydro-23-oxoolean-12-en-28-oic C30H46O5 (Tava et al., acid 2011)

Medicagenic acid C30H46O6

Zanhic acid C30H46O7

Oleanolic acid; Soyasapogenol E C30H48O3

Hederagenin, 2-hydroxyoleanolic acid; C30H48O4

Queretaroic acid

Bayogenin; 2-hydroxyqueretaroic acid; C30H48O5

Caulophyllogenin

Sophoradiol; 3,24-dihydroolean-12-ene C30H50O2

Soyasapogenol B C30H50O3

Soyasapogenol A C30H50O4

Panax Dammarenediol C30H52O2 (Zou et al.,

Panaxadiol; Protopanaxadiol C30H52O3 2002)

Panaxatriol; Protopanaxatriol C30H52O4

Bupleurum Rotundioside O sapogenin C30H46O4 (Ashour and

Rotundioside L, M sapogenin C30H46O5 Wink, 2011) Sandrosapogenin III, VIII C30H46O6

Sandrosapogenin IV C30H46O7

Saikogenin C, M; Rotundioside B, C C30H48O3

sapogenin; Sondrosapogenin IX, X;

Rotundifolioside A, I, J sapogenin

Saikogenin A, Bl, B2, D, G, K, N, O, P, S; C30H48O4

Prosaikogenin A, H; Rotundioside A, J, K,

Q, S, V sapogenin; Rotundifolioside D, E, F,

G, H

Sandrosapogenin II, V, VI; Bupleuroside VI C30H48O5

sapogenin; Saikosapogenin L, Q, Q2, R, U,

V2, V; Scorzoneroside A, B, C sapogenin

Rotundioside D sapogenin 30H50O

Rotundifolioside B, C 30H50O

Hydroxysaikosapogenin A, C, D; 30H50O

Bupleuroside VIII sapogenin

Rotundioside N sapogenin 31H50O

Saikosapogenin F 31H52O

Methoxysaikosapogenin F; H52O

Saikosapogenin T; Bupleuroside IX

sapogenin; Rotundioside X, Y sapogenin

Saikogenin B3, B4; Rotundioside P, R, U 31H52O

sapogenin

Maesa 16-oxo-28-hydroxyolean-12-ene 3oH ₄ sO (Manguro et

16,22- dihydroxyolean-12-en-28-al 3oH ₄ gO al., 2011) 16,21,22-trihydroxyoleanane-13:28-ollide 3oH ₄ gO

16,28-dihydroxyolean-12-ene 30H50O

16,22,28-trihydroxyolean-12-ene 30H50O Sapogenins comprised in commonly Chemical formula Reference accumulating saponins of sapogenin

16,21,22,28-tetrahydroxyolean-12-ene C30H50O5

Maesasapogenin I, I I, I II, IV, V, VI, VII C30H50O6

Quillaic acid C30H46O5 (Guo et al.,

1998)

Betulin C30H48O2 ( ickling and

Betulinic acid C30H48O3 Glombitza,

Betulafolientetraol C30H52O5 1993)

Oleanolic acid; Ursolic acid C30H48O3 (Stiti et al.,

Maslinic acid C30H48O4 2007)

Bacchara-12,21-dien-3-ol C3oH ₄₈ 0

Butyrospermol C30H50O

The method of the present invention makes use of cyclodextrins for the production of triterpenoid sapogenins in the culture medium of eukaryotic cells that are capable of synthesizing triterpenoid sapogenins. With "cyclodextrins" (CDs) (sometimes also called cycloamyloses) are meant cyclic oligosaccharides composed of 5 or more (a-l,4)-linked a-D-glucopyranose subunits, which are well- known in the art. As used herein, "cyclodextrins" encompass both naturally occurring cyclodextrins as well as chemical derivatives thereof, as described further herein. Cyclodextrins possess a cage-like supramolecular structure, and are capable of forming inclusion complexes with a variety of guest molecules: CDs incorporate compounds in their hydrophobic cavities depending on the cavity size. The most typical cyclodextrins contain a set of 6 to 8 glucopyranoside units in a ring (the cyclodextrin core), creating a cone shape. Within this family, a-cyclodextrins (aCD) have 6 glucopyranoside units, β- cyclodextrins ( CD) have 7 glucopyranoside units, and γ-cyclodextrins (yCD) have 8 glucopyranoside units in a ring. Each glucopyranoside unit has, according to the standard atom numbering system, one primary alcohol group at carbon 6 and two secondary alcohol groups at carbons 2 and 3. These natural cyclodextrins, in particular CD, are of limited aqueous solubility. Therefore, several derivatives of cyclodextrins have been developed. Numerous chemical modifications of cyclodextrins are known in the art, as summarized for instance by A. Croft and R. Bartsch in Tetrahedron Report No. 147, Tetrahedron (1983) 39(9):1417-1474, and which are incorporated herein by reference. These derivatives usually are produced by aminations, esterifications or etherifications of primary and secondary hydroxyl groups of the cyclodextrins. Depending on the substituent, the solubility of the cyclodextrin derivatives is usually different from that of their parent cyclodextrins. Virtually all derivatives have a changed hydrophobic cavity volume and also these modifications can improve solubility, stability against light or oxygen and help control the chemical activity of guest molecules. For example, and without the purpose of being limitative, water-soluble cyclodextrin derivatives of commercial interest include the hydroxypropyl derivatives of CD and yCD, the randomly methylated β-cyclodextrin (RM CD), and sulfobutylether β-cyclodextrin sodium salt (SBE CD). Thus, according to a preferred embodiment, the cyclodextrin that is used in the method of the present invention is chosen from the group comprising randomly methylated cyclodextrin or hydroxypropylated cyclodextrin. Preferably, the degree of substitution by methyls per glucose unit of the randomly methylated cyclodextrin is between 1 and 3, and more preferably, the degree of substitution by methyls per glucose unit of the randomly methylated cyclodextrin is 2. Preferably, the degree of substitution by hydroxypropyls per glucose unit of the hydroxypropylated cyclodextrin is between 0.6 and 0.9. According to another preferred embodiment, the cyclodextrin that is used in the method of the present invention is a β-cyclodextrin. According to still another prefered embodiment, the cyclodextrin that is used in the method of the present invention is a methylated β-cyclodextrin.

In another preferred embodiment, the concentration of cyclodextrins in the culture medium is less than 25 mM, preferably less than 10 mM, more preferably between 2 and 7 mM. According to more specific embodiments, the concentration of cyclodextrins in the culture medium is 5 mM or 2.5 mM, or 1 mM. In one other embodiment, cyclodextrins are added to the culture medium at one point in time, for example immediately before or after inoculation with eukaryotic cells. Preferably, cyclodextrins are added to the culture medium at different consecutive time points, for example immediately before or after inoculation with eukaryotic cells, and then on a daily basis after the first addition of cyclodextrins, or every other day after the first addition of cyclodextrins. This is further illustrated, without the purpose of being limitative, in Example 5. With "production" of triterpenoid sapogenins is meant both intracellular production as well as secretion into the medium. According to a preferred embodiment, the triterpenoid sapogenins as produced by the method of the present invention are secreted into the extracellular medium. The production of triterpenoid sapogenins typically is enhanced or induced by using the method of the invention. An "enhanced production" of a triterpenoid sapogenin means that there exists already a detectable amount of this metabolite in the eukaryotic cell in the absence of cyclodextrins but that detection only becomes possible in the extracellular medium upon adding cyclodextrins according to the invention. An "induced production" of a triterpenoid sapogenin means that there is no detectable production of this metabolite in the eukaryotic cell in the absence of cyclodextrins but that detection becomes possible in the cell and the extracellular medium upon addition of cyclodextrins according to the invention. With an increase in the production of one or more sapogenins according to the method of the invention, it is understood that said production may be enhanced or induced with a factor 2, 3, 4, 5, 10, 20, 50, 100 or more, relative to the production in the absence of cyclodextrins. Eukaryotic cells provided in the above described method can be of any unicellular or multicellular eukaryotic organism, but in particular embodiments microbial, plant, and algal cells are envisaged. The nature of the cells used will typically depend on the desired sapogenins and/or the ease and cost of producing the sapogenins. According to particular embodiments, the plant cell as used is derived from a plant of the genus selected from the group comprising Medicago, Panax, Bupleurum, Maesa, Saponaria, Betula, Quillaja, Aesculus, Chenopodium, Hedera, Acacia, Centella, Oleander, Avena, Arabidopsis, or Nicotiana. The term "plant" as used herein refers to vascular plants (e.g. gymnosperms and angiosperms). According to further particular embodiments, the microbial cell as used is a yeast cell, in particular a yeast cell of a Saccharomyces species (e.g. Saccharomyces cerevisiae), a Hansenula species (e.g. Hansenula polymorpha), a Yarrowia species (e.g. Yarrowia lipolytica), a Kluyveromyces species (e.g. Kluyveromyces lactis), a Pichia species (e.g. Pichia pastoris) or a Candida species (e.g. Candida utilis). According to a specific embodiment, the eukaryotic cells as used are Saccharomyces cells. According to further particular embodiments, the algal cells are derived from algae of the genus selected from the group comprising Dunaliella, Chlorella, or Chlamydomonas. According to a very particular embodiment, the eukaryotic cells as used are not plant cells.

In a particular embodiment, the eukaryotic cells may naturally have the capability of synthesizing triterpene saponins and triterpene sapogenin building blocks, such as plant cells. A "plant cell" is understood, according to the invention, as being any cell which is derived from or found in a plant and which is able to form or is part of undifferentiated tissues, such as call i or cell cultures, differentiated tissues such as embryos, parts of plants, plants or seeds.

In an alternative embodiment, the eukaryotic cells as used herein themselves do not naturally produce triterpenoid sapo(ge)nins, but may do so after genetic engineering. Thus, preferably, eukaryotic cells artificially producing sapogenins refers to cells that, while not naturally having the ability to synthesize sapogenins, have acquired such ability by means of genetic manipulation processes including transgenesis. This particularly applies to yeast cells or algal cells, which naturally do not synthesize sapogenins. In yet another embodiment, the eukaryotic cells as used may be genetically engineered to produce another spectrum of sapogenins, as compared to the natural spectrum that is produced by the wild type strain, which particularly may apply to plant cells.

Thus, according to a preferred embodiment, the plant cell as used may be a genetically engineered plant cell, which is a plant cell derived from a transgenic plant. A "transgenic plant", as used herein, refers to a plant comprising a recombinant polynucleotide and/or a recombinant polypeptide resulting in the expression of a regulatory or biosynthetic enzyme of the sapogenin biosynthesis pathway. A transgenic plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, and progeny thereof. A transgenic plant can be obtained by transforming a plant cell with an expression cassette and regenerating such plant cell into a transgenic plant. Such plants can be propagated vegetatively or reproductively. The transforming step may be carried out by any suitable means, including by Agrobacterium-mediated transformation and non-Agrobactem/m-mediated transformation, as discussed further below. Plants can be regenerated from the transformed cell (or cells) by techniques known to those skilled in the art. Where chimeric plants are produced by the process, plants in which all cells are transformed may be regenerated from chimeric plants having transformed germ cells, as is known in the art. Methods that can be used to transform plant cells or tissue with expression vectors include both Agrobacterium and non- Agrobacterium vectors. Agrobacter um-mediated gene transfer exploits the natural ability of Agrobacterium tumefaciens to transfer DNA into plant chromosomes and is described in detail in Gheysen, G., Angenon, G. and Van Montagu, M. 1998. Agrobacterium-mediated plant transformation: a scientifically intriguing story with significant applications. In K. Lindsey (Ed.), Transgenic Plant Research. Harwood Academic Publishers, Amsterdam, pp. 1-33 and in Stafford, H.A. (2000) Botanical Review 66: 99-118. A second group of transformation methods is the non-Agrobacterium mediated transformation and these methods are known as direct gene transfer methods. An overview is brought by Barcelo, P. and Lazzeri, P. A. (1998) Direct gene transfer: chemical, electrical and physical methods. In K. Lindsey (Ed.), Transgenic Plant Research, Harwood Academic Publishers, Amsterdam, pp.35-55. Methods include particle gun delivery, microinjection, electroporation of intact cells, polyethyleneglycol-mediated protoplast transformation, electroporation of protoplasts, liposome- mediated transformation, silicon-whiskers mediated transformation etc. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line (wild type) used to generate a transgenic plant herein. Genetically transformed hairy root cultures can be obtained by transformation with virulent strains of Agrobacterium rhizogenes, and they can produce high contents of secondary metabolites, including triterpenoid sapo(ge)nins, characteristic to the mother plant. Protocols used for establishing of hairy root cultures vary, as well as the susceptibility of plant species to infection by Agrobacterium (Toivounen et al. 1993; Vanhala et al. 1995). It is known that the Agrobacterium strain used for transformation has a great influence on root morphology and the degree of secondary metabolite accumulation in hairy root cultures. It is possible by systematic clone selection e.g. via protoplasts, to find high yielding, stable, and from single cell derived-hairy root clones. This is possible because the hairy root cultures possess a great somaclonal variation. Another possibility of transformation is the use of viral vectors (Turpen 1999). Any plant tissue or plant cells capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with an expression vector of interest. The term 'organogenesis' means a process by which shoots and roots are developed sequentially from meristematic centers; the term 'embryogenesis' means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include protoplasts, leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g. apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyls meristem).

According to a particularly preferred embodiment, the yeast cell as used in any of the above described methods is a genetically engineered yeast cell, such as a yeast cell expressing an exogenous regulatory or biosynthetic enzyme of the sapogenin biosynthesis pathway (see also Table 2 and 3). Preferably, the genetically engineered yeast cell is overexpressing an oxidosqualene cyclase (EC 5.4.99.-) and/or a cytochrome P450 (EC 1.14.-). According to a particular embodiment, the genetically engineered yeast cell as used in any of the above described methods is overexpressing a cytochrome P450 which is capable of hydroxylating a carbon at position 21 of an oleanane-type backbone as pictured in Figure 10. According to a preferred embodiment, the genetically engineered yeast cell as used in any of the above described methods is overexpressing SEQ ID NO: 3 or 4, or fragments or variants thereof (as described further herein).

According to a further preferred embodiment, the genetically engineered yeast cell as used is deficient in expression and/or activity of an enzyme involved in endogenous sterol synthesis. For example, and without the purpose of being limitative, key enzymes in the yeast ergosterol biosynthetic pathway may be downregulated, in particular the lanosterol synthase gene (EC 5.4.99.7) to enable maximal and controllable flux towards the heterologous production of the desired triterpenoid sapogenin compounds.

The term "endogenous" as used herein, refers to substances (e.g. genes) originating from within an organism, tissue, or cell. Analogously, "exogenous" as used herein is any material originated outside of an organism, tissue, or cell, but that is present (and typically can become active) in that organism, tissue, or cell. Table 2. Overview of oxidosqualene cyclase (OSCs) reported in the literature as involved in sapogenin biosynthesis. 2,3-Oxidosqualene cyclization products identified to emerge from the activity of the corresponding OSC are indicated in squared brackets: aa - a-amyrin, ba - β-amyrin, bau - baurenol, da - damyrin, dam - dammarenediol, fri - friedelin, ge - germanicol, glu - glutinol, isotir - isotirucallol, lu - lupeol, lud - lupane-3 ,20-diol, tir - tirucalla-7,24-dien-3 -ol, minor - additional byproducts either reported to be of minor appearance or to represent <10% of the observed products (Table derived from Augustin et al. 2011).

GenBank

Name ID Plant species Product

Accurate b-amyrin synthases

AaBAS ACA13386 A.annua [ba]

AsOXAl AAX14716 A.sedifolius [ba]

AsbASl CAC84558 A.strigosa [ba]

BgbAS BAF80443 B.gymnorhiza [ba]

BPY BAB83088 B.platyphylla [ba]

EtAS BAE43642 E.tirucalli [ba]

GgbASl BAA89815 G.glabra [ba]

GsASl AC024697 G.straminea [ba]

cOSCl BAE53429 Ljaponicus [ba]

b-AS = MtAMYl CAD23247 M.truncatula [ba]

NsbASl ACH88048 N.sativa [ba]

PNY BAA33461 P.ginseng [ba]

PNY2 BAA33722 P.ginseng [ba]

PSY BAA97558 P.sativum [ba]

SITTS1 ADU52574 S.lycopersicum [ba]

SvBS ABK76265 S.vaccaria [ba]

Accurate lupeol synthases

BgLUS BAF80444 B.gymnorhiza [lu]

BPW BAB83087 B.platyphylla [lu]

GgLUSl BAD08587 G.glabra [lu]

cOSC3 BAE53430 Ljaponicus [lu]

OEW BAA86930 O.europaea [lu]

RcLUS ABB76766 R.communis [lu]

TRW BAA86932 T.officinale [lu]

Accurate dammarenediol

synthases

CaDDS AAS01523 C.asiatica [dam]

PNA = DDS BAF33291 P.ginseng [dam]

DDS = PNA ACZ71036 P.ginseng [dam]

Multifunctional OSCs

LUPl/Atlg78970 NP_178018 A.thaliana [lu/lud +4 minor]

LUP5/Atlg66960 NP_176868 A.thaliana [tir/isotir +4

PEN6/Atlg78500 NP_177971 A.thaliana [lu/bau/aa +5

CsOSC2/CSV BAB83254 C.speciosus [ba/ge/lu +add. GenBank

Name ID Plant species Product

LjAMY2 AAO33580 L.japonicus [ba/lu +1 minor]

KcMS BAF35580 K.candel [lu/ba/aa]

KdGLS ADK35124 K. daigremontiana [glu/fri/ba +1

OEA BAF63702 O.europaea [aa/ba +3minor]

PSM BAA97559 P. sativum [aa/ba +6minor]

RsMl BAF80441 R.stylosa [ge/ba +1 minor]

[da/aa/ba +4

SITTS2 ADU52575 S.lycopersicum minor]

Table 3. Overview of CYPs reported in the literature as involved in sapogenin biosynthesis

Name GenBank ID Plant species Reference

CYP51H 10 ABG88965 A. strigosa (Kunii et al., 2012)

CYP716A12 FN995113 M. truncatula (Carelli et al., 2011)

CYP716A15 BAJ84106 V. vinifera (Fukushima et al.,

2011)

CYP716A17 BAJ84107 V. vinifera (Fukushima et al.,

2011)

CYP716AL1 FN995113 C. roseus (Huang et al., 2012)

CYP716A47 AEY75212 P. ginseng (Han et al., 2011)

CYP716A53v2 AFO63031 P. ginseng (Han et al., 2012)

CYP72A61v2 BAL45199 M. truncatula (Fukushima et al.,

2013)

CYP72A63 AB558146 M. truncatula (Seki et al., 2011)

CYP72A68v2 BAL45204 M. truncatula (Fukushima et al.,

2013)

CYP72A154 AB558153 G. uralensis (Seki et al., 2011)

CYP88D6 AB433179 G. uralensis (Seki et al., 2008)

CYP93E1 N M_001249225 G. max (Shibuya et al., 2006)

CYP93E2 DQ335790 M. truncatula (Li et al., 2007)

CYP93E3 AB437320 G. uralensis (Seki et al., 2008)

Suitable cell culture media for eukaryotic cells, in particular plant cells and microbial cells, are known in the art. For plant cells, exemplary media include standard growth media, many of which are commercially available (e.g., Sigma Chemical Co., St. Louis, Mo.). Examples include Schenk-Hildebrandt

(SH) medium, Linsmaier-Skoog (LS) medium, Murashige and Skoog (MS) medium, Gamborg's B5 medium, Nitsch & Nitsch medium, White's medium, and other variations and supplements well known to those of skill in the art (see, e.g., Plant Cell Culture, Dixon, ed. I RL Press, Ltd. Oxford (1985) and George et al., Plant Culture Media, Vol 1, Formulations and Uses Exegetics Ltd. Wilts, UK, (1987)). (see, e.g., Plant Cell Culture, Dixon, ed. I RL Press, Ltd. Oxford (1985) and George et al., Plant Culture Media,

Vol 1, Formulations and Uses Exegetics Ltd. Wilts, U K, (1987)). For yeast cells, exemplary media include standard growth media, many of which are commercially available (e.g., Clontech, Sigma Chemical Co., St. Louis, Mo.). Examples include Yeast Extract Peptone Dextrose (YPD or YPED) medium, Yeast Extract Peptone Glycerol (YPG or YPEG) medium, Hartwell's complete (HC) medium, Synthetic complete (SC) medium, Yeast Nitrogen Base (YNB), and other variations and supplements well known to those of skill in the art (see, Yeast Protocol Handbook, Clontech).

The incubation conditions (temperature, photoperiod, shaking, auxin/cytokine hormone ratio, promoter inducing conditions, promoter repressing conditions, etc.) will depend, among other factors, on the cells to be incubated and are standard techniques in the art. In a particular embodiment, the current invention can be combined with other known methods to enhance the production and/or the secretion of triterpenoid sapogenin production in eukaryotic cell cultures, for example (1) by improvement of the cell culture conditions, (2) by metabolic engineering, (3) by the addition of specific elicitors to the cell culture.

Preferably, the eukaryotic cell is induced before it produces secondary metabolites such as triterpenoid sapogenins, meaning that the cell culture is stimulated by the addition of an external factor. External factors include the application of heat, the application of cold, the addition of acids, bases, metal ions, fungal membrane proteins, sugars and the like. In the case of plants, it is demonstrated that better production of plant secondary metabolites occurs via elicitation. Elicitors are compounds capable of inducing defense responses in plants. These are usually not found in intact plants but their biosynthesis is induced after wounding or stress conditions. Commonly used elicitors are jasmonates, mainly jasmonic acid and its methyl ester, methyl jasmonate. Jasmonates are linoleic acid derivatives of the plasma membrane and display a wide distribution in the plant kingdom. They were originally classified as growth inhibitors or promoters of senescence but now it has become apparent that they have pleiotropic effects on plant growth and development. Jasmonates appear to regulate cell division, cell elongation and cell expansion and thereby stimulate organ or tissue formation. They are also involved in the signal transduction cascades that are activated by stress situations such as wounding, osmotic stress, desiccation and pathogen attack. Methyl jasmonate (MeJA) is known to induce the accumulation of numerous defense-related secondary metabolites through the induction of genes coding for the enzymes involved in the biosynthesis of these compounds in plants. Jasmonates can modulate gene expression from the (post)transcriptional to the (post)translational level, both in a positive as well as in a negative way. Genes that are upregulated are e.g. defense and stress related genes (PR proteins and enzymes involved with the synthesis of phytoalexins and other secondary metabolites) whereas the activity of housekeeping proteins and genes involved with photosynthetic carbon assimilation are down- regulated. For example: the biosynthesis of phytoalexins and other secondary products in plants can also be boosted up by signal molecules derived from micro-organisms or plants (such as peptides, oligosaccharides, glycopeptides, salicylic acid and lipophilic substances) as well as by various abiotic elicitors like UV-light, heavy metals (Cu, VOS04, Cd) and ethylene. The effect of any elicitor is dependent on a number of factors, such as the specificity of an elicitor, elicitor concentration, the duration of the treatment and growth stage of the culture.

A number of suitable culture media for callus induction and subsequent growth on aqueous or solidified media are known. Exemplary media include standard growth media, many of which are commercially available (e.g., Sigma Chemical Co., St. Louis, Mo.). Examples include Schenk-Hildebrandt (SH) medium, Linsmaier-Skoog (LS) medium, Murashige and Skoog (MS) medium, Gamborg's B5 medium, Nitsch & Nitsch medium, White's medium, and other variations and supplements well known to those of skill in the art

During or after the sapogenin production in the growth medium of a eukaryotic cell culture according to any of the above described methods, the sapogenins can be extracted from the cells. According to a preferred embodiment, the sapogenins are extracted from the culture medium wherein the sapogenins are secreted. Accordingly, the methods of producing sapogenins may optionally also comprise the step of extracting the sapogenins from the culture medium. Eventually, the sapogenins may also be further purified. Means that may be employed to this end are known to the skilled person. Generally, triterpenoid sapogenins can be measured intracellularly or in the extracellular space by methods known in the art. Such methods comprise analysis by thin-layer chromatography, high pressure liquid chromatography, capillary electrophoresis, gas chromatography combined with mass spectrometric detection, radioimmuno-assay ( IA) and enzyme immuno-assay (ELISA). For example, Medicago triterpene sapo(ge)nin content can be analysed by Reversed phase UPLC/ICR/FT-MS, and is also further illustrated in the Example section. In a further aspect, the invention also provides a eukaryotic cell genetically engineered to synthesize sapogenins and/or pathway intermediates. In particular, the genetically engineered cell is a yeast cell, for example a Saccharomyces, Schizosaccharomyces, Pichia, Yarrowia, Candida or Hansenula cell. According to a particularly preferred embodiment, the yeast cell is genetically engineered to express an exogenous regulatory or biosynthetic enzyme of the sapogenin biosynthesis pathway (see also Table 2 and 3). Preferably, the yeast cell is genetically engineered to overexpress an oxidosqualene cyclase (EC 5.4.99.-) and/or a cytochrome P450 (EC 1.14.-). According to a particular embodiment, the yeast cell is genetically engineered to overexpress a cytochrome P450 which is capable of hydroxylating a carbon at position 21 of an oleanane-type backbone as pictured in Figure 10. According to a preferred embodiment, the yeast cell is genetically engineered to overexpress SEQ ID NO: 3 or 4, or fragments or variants thereof (as described further herein).

In still another aspect, the present invention also encompasses existing or novel sapogenins obtained by any of the above described methods. The sapogenins that are extracellularly accumulating in the growth medium of a eukaryotic cell culture in the presence of cyclodextrins are readily accessible and can be exploited by industry for a variety of purposes, either directly, or after further synthetic chemistry. For examples, sapogenins and its derivatives (including saponins) can be used as additives to foods and cosmetics, preservatives, flavor modifiers, agents for removal of cholesterol from dietary products, and may also be very valuable for their pharmacological properties. For example, several saponins and sapogenins are considered to possess activities such as anti-inflammatory, anti- carcinogenic, anti-bacterial, anti-fungal and anti-viral effects. Saponins are also of interest as valuable adjuvants and the first saponin-based vaccines are introduced commercially (reviewed in Sun et al. 2009). In a specific embodiment, sapogenins that are hydroxylated on a carbon at position 21 of an oleanane- type backbone as pictured in Figure 10 are encompassed here.

According to another aspect, the invention relates to an isolated polypeptide selected from the group consisting of:

(a) a polypeptide encoded by a polynucleotide comprising SEQ ID NO: 1 or 2;

(b) a polypeptide comprising a polypeptide sequence having a least 75% identity to the polypeptide encoded by a polynucleotide sequence having SEQ ID NO: 1 or 2;

(c) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3 or 4;

(d) a polypeptide comprising an amino acid sequence with at least 75% identity to SEQ ID NO: 3 or 4;

(e) fragments and/or variants of the polypeptides according to (a), (b), (c), (d).

In one embodiment, the invention relates to any of the above described polypeptides wherein said polypeptide sequence is consisting of an amino acid sequence as set forth in SEQ ID NO: 3 or 4 and polypeptide sequences having at least 75% identity to SEQ ID NO: 3 or 4.

According to another aspect, the invention relates to an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a polynucleotide sequence having the sequence SEQ ID NO: 1 or 2;

(b) a polynucleotide comprising a polynucleotide sequence having at least 70% identity to the sequence having SEQ ID NO: 1 or 2;

(c) a polynucleotide which encodes the polypeptide sequence as set forth in SEQ ID NO: 3 or 4; (d) a polynucleotide which encodes the polypeptide sequence as set forth in SEQ ID NO: 3 or 4;

(e) fragments and variants of the polynucleotides according to (a), (b), (c) or (d).

As used herein, the terms "polypeptide", "protein", "peptide" are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

As used herein, the terms "nucleic acid", "polynucleotide", "polynucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger NA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotide molecule may be linear or circular. The polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like. The polynucleotide may comprise single stranded or double stranded DNA or RNA. The polynucleotide may comprise modified bases or a modified backbone. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide.

An "isolated polypeptide" or an "isolated polynucleotide", as used herein, refers to respectively an amino acid sequence or a polynucleotide sequence which is not naturally-occurring or no longer occurring in the natural environment wherein it was originally present.

As used herein, the terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 75% identity over a specified region) when compared and aligned for maximum correspondence over a comparison window or designated region as measured using sequence comparison algorithms or by manual alignment and visual inspection. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides or even more in length. According to preferred embodiments, the invention relates to an isolated polypeptide comprising a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the polypeptide encoded by a polynucleotide sequence having SEQ ID NO: 1 or 2, or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:3 or 4. In other preferred embodiments, the invention relates to an isolated polynucleotide comprising a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence having SEQ ID NO: l or 2.

In a particular embodiment, fragments and variants of any of the above polynucleotides or polypeptides also form part of the present invention.

In reference to a nucleotide sequence "a fragment" refers to any sequence of at least 15 consecutive nucleotides, preferably at least 30 consecutive nucleotides, more preferably at least 50, 60, 70, 80, 90, 100, 150, 200 consecutive nucleotides or more, of any of the sequences provided herein. If desired, the fragment may be fused at either terminus to additional base pairs, which may number from 1 to 20, typically 50 to 100, but up to 250 to 500 or more.

A "fragment", as referred to polypeptides, refers to a subsequence of the polypeptide. Fragments may vary in size from as few as 5 amino acids to the length of the intact polypeptide, but are preferably at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75 amino acids in length. If desired, the fragment may be fused at either terminus to additional amino acids, which may number from 1 to 20, typically 50 to 100, but up to 250 to 500 or more. A "functional fragment" means a polypeptide fragment possessing the ability to hydroxylate a carbon at position 21 of an oleanane-type backbone as pictured in Figure 10.

A "variant" as used herein refers to homologs, orthologs and paralogs and include, but are not limited to, homologs, orthologs and paralogs of SEQ ID NOs: 1-4. Homologs of a protein encompass peptides, oligopeptides and polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived; or in other words, without significant loss of function or activity. Orthologs and paralogs, which are well-known terms by the skilled person, define subcategories of homologs and encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that have originated through duplication of an ancestral gene; orthologs are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologues and paralogues include phylogenetic methods, sequence similarity and hybridization methods. Percentage similarity and identity can be determined electronically. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Preferably, said homologue, orthologue or paralogue has a sequence identity at protein level of at least 50%, preferably 60%, more preferably 70%, even more preferably 80%, most preferably 90% as measured in a BLASTp.

Further, it will be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the polypeptides of the present invention. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides. Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed "silent" variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence are a feature of the invention. In addition to silent variations, other conservative variations that alter one, or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide (i.e. enhanced secondary metabolite production, in the context of the present invention), these conservative variants are, likewise, a feature of the invention. Conservative substitutions or variations, as used herin, are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table depicted further herein. This table shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions. Residue Conservative Substitutions

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Gin Asn

Cys Ser

Glu Asp

Gly Pro

His Asn, Gin

lie Leu, Val

Leu lie, Val

Lys Arg, Gin

Met Leu, lie

Phe Met, Leu, Tyr

Ser Thr, Gly

Thr Ser, Val

Trp Tyr

Tyr Trp, Phe

Val lie, Leu

Substitutions that are less conservative than those in the above Table can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. Substitutions, deletions and insertions introduced into the sequences are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis or the other methods known in the art. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotides of the invention should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function (i.e. hydroxylating a carbon at position 21 of an oleanane-type backbone as pictured in Figure 10, in the context of the present invention).

In a specific embodiment, the invention relates to a chimeric gene comprising the following operably- linked sequences: a) a promoter region capable of directing expression in a eukaryotic cell (as defined herein before); b) a DNA region encoding a polypeptide as defined above; c) a 3' polyadenylation and transcript termination region.

The term "operably linked" as used herein refers to a linkage in which the regulatory sequence is contiguous with the gene of interest to control the gene of interest, as well as regulatory sequences that act in trans or at a distance to control the gene of interest. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter and allows transcription elongation to proceed through the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if it is expressed as a pre-protein that participates in the transport of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or adapters or linkers inserted in lieu thereof using restriction endonucleases known to one of skill in the art.

The term "regulatory sequence" as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operably linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRMA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

A vector comprising a polynucleotide sequence or a chimeric gene as defined above also forms part of the present invention, as well as a host cell comprising a polynucleotide sequence or a chimeric gene or a vector as defined above.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. The vector may be of any suitable type including, but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid or even an artificial chromosome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of certain genes of interest. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired and according to a particular host organism (e.g. plant cell). Typically, a recombinant vector according to the present invention comprises at least one "chimeric gene" or "expression cassette". Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof of the present invention operably linked with the transcription initiation region, and a termination sequence including a stop signal for NA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as plant cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell.

The term "recombinant host cell" ("expression host cell", "expression host system", "expression system" or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. Host cells can be of bacterial, fungal, plant or mammalian origin.

According to yet another aspect, the invention provides a transgenic plant or a cell derived thereof that is transformed with the above described vector.

The term "plant" as used herein refers to vascular plants (e.g. gymnosperms and angiosperms). A "transgenic plant" refers to a plant comprising a recombinant polynucleotide and/or a recombinant polypeptide according to the invention. A transgenic plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, and progeny thereof. A transgenic plant can be obtained by transforming a plant cell with an expression cassette of the present invention and regenerating such plant cell into a transgenic plant. Such plants can be propagated vegetatively or reproductively. The transforming step may be carried out by any suitable means, including by Agrobacterium-mediated transformation and non-Agrobacter/ ^' um-mediated transformation, as discussed in detail below. Plants can be regenerated from the transformed cell (or cells) by techniques known to those skilled in the art. Where chimeric plants are produced by the process, plants in which all cells are transformed may be regenerated from chimeric plants having transformed germ cells, as is known in the art. Methods that can be used to transform plant cells or tissue with expression vectors of the present invention include both Agrobacterium and non- Agrobacterium vectors. Agrobacterium-mediated gene transfer exploits the natural ability of Agrobacterium tumefaciens to transfer DNA into plant chromosomes and is described in detail in Gheysen, G., Angenon, G. and Van Montagu, M. 1998. Agrobacterium-mediated plant transformation: a scientifically intriguing story with significant applications. In K. Lindsey (Ed.), Transgenic Plant Research. Harwood Academic Publishers, Amsterdam, pp. 1-33 and in Stafford, H.A. (2000) Botanical Review 66: 99-118. A second group of transformation methods is the non-Agrobacterium mediated transformation and these methods are known as direct gene transfer methods. An overview is brought by Barcelo, P. and Lazzeri, P. A. (1998) Direct gene transfer: chemical, electrical and physical methods. In K. Lindsey (Ed.), Transgenic Plant Research, Harwood Academic Publishers, Amsterdam, pp.35-55. Methods include particle gun delivery, microinjection, electroporation of intact cells, polyethyleneglycol-mediated protoplast transformation, electroporation of protoplasts, liposome- mediated transformation, silicon-whiskers mediated transformation etc. Hairy root cultures can be obtained by transformation with virulent strains of Agrobacterium rhizogenes, and they can produce high contents of secondary metabolites characteristic to the mother plant. Protocols used for establishing of hairy root cultures vary, as well as the susceptibility of plant species to infection b Agrobacterium (Toivounen et al. 1993; Vanhala et al. 1995). It is known that the Agrobacterium strain used for transformation has a great influence on root morphology and the degree of secondary metabolite accumulation in hairy root cultures. It is possible by systematic clone selection e.g. via protoplasts, to find high yielding, stable, and from single cell derived-hairy root clones. This is possible because the hairy root cultures possess a great somaclonal variation. Another possibility of transformation is the use of viral vectors (Turpen 1999). Any plant tissue or plant cells capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with an expression vector of the present invention. The term 'organogenesis' means a process by which shoots and roots are developed sequentially from meristematic centers; the term 'embryogenesis' means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include protoplasts, leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g. apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyls meristem). A "control plant" as used in the present invention refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying a difference in production of sapogenins in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line (wild type) used to generate a transgenic plant herein.

Plants of the present invention may include, but not limited to, plants or plant cells of agronomically important crops which are or are not intended for animal or human nutrition, such as maize or corn, wheat, barley, oat, Brassica spp. plants such as Brassica napus or Brassica juncea, soybean, bean, alfalfa, pea, rice, sugarcane, beetroot, tobacco, sunflower, cotton, Arabidopsis, vegetable plants such as cucumber, leek, carrot, tomato, lettuce, peppers, melon, watermelon, diverse herbs such as oregano, basilicum and mint. It may also be applied to plants that produce valuable compounds, e.g. useful as for instance pharmaceuticals, as ajmalicine, vinblastine, vincristine, ajmaline, reserpine, rescinnamine, camptothecine, ellipticine, quinine, and quinidine, taxol, morphine, scopolamine, atropine, cocaine, sanguinarine, codeine, genistein, daidzein, digoxin, calystegins or as food additives such as anthocyanins, vanillin; including but not limited to the classes of compounds mentioned above. Examples of such plants include, but not limited to, Papaver spp., Rauwolfia spp., Taxus spp., Cinchona spp., Eschscholtzia californica, Camptotheca acuminata, Hyoscyamus spp., Berberis spp., Coptis spp., Datura spp., Atropa spp., Thalictrum spp., Peganum spp. Preferred members of the genus Taxus comprise Taxus brevifolia, Taxus baccata, Taxus cuspidata, Taxus canadensis and Taxus floridana.

The polynucleotide sequence, homologue, variant or fragment thereof of the invention may be expressed in for example a plant cell under the control of a promoter that directs constitutive expression or regulated expression. Regulated expression comprises temporally or spatially regulated expression and any other form of inducible or repressible expression. Temporally means that the expression is induced at a certain time point, for instance, when a certain growth rate of the plant cell culture is obtained (e.g. the promoter is induced only in the stationary phase or at a certain stage of development). Spatially means that the promoter is only active in specific organs, tissues, or cells (e.g. only in roots, leaves, epidermis, guard cells or the like). Other examples of regulated expression comprise promoters whose activity is induced or repressed by adding chemical or physical stimuli to the plant cell. In a preferred embodiment the expression is under control of environmental, hormonal, chemical, and/or developmental signals. Such promoters for plant cells include promoters that are regulated by (1) heat, (2) light, (3) hormones, such as abscisic acid and methyl jasmonate (4) wounding or (5) chemicals such as salicylic acid, chitosans or metals. Indeed, it is well known that the expression of secondary metabolites can be boosted by the addition of for example specific chemicals, jasmonate and elicitors. In a particular embodiment the co-expression of several (more than one) polynucleotide sequence or homologue or variant or fragment thereof, in combination with the induction of secondary metabolite synthesis is beneficial for an optimal and enhanced production of secondary metabolites. Alternatively, the at least one polynucleotide sequence, homologue, variant or fragment thereof is placed under the control of a constitutive promoter. A constitutive promoter directs expression in a wide range of cells under a wide range of conditions. Examples of constitutive plant promoters useful for expressing heterologous polypeptides in plant cells include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues including monocots; the nopaline synthase promoter and the octopine synthase promoter. The expression cassette is usually provided in a DNA or RNA construct which is typically called an "expression vector" which is any genetic element, e.g., a plasmid, a chromosome, a virus, behaving either as an autonomous unit of polynucleotide replication within a cell (i.e. capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, bacteriophages, cosmids, plant viruses and artificial chromosomes. The expression cassette may be provided in a DNA construct which also has at least one replication system. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. The markers may a) code for protection against a biocide, such as antibiotics, toxins, heavy metals, certain sugars or the like; b) provide complementation, by imparting prototrophy to an auxotrophic host: or c) provide a visible phenotype through the production of a novel compound in the plant. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non- limiting examples of suitable markers are β-glucuronidase, providing indigo production, luciferase, providing visible light production, Green Fluorescent Protein and variants thereof, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.

The term "promoter activity" refers to the extent of transcription of a polynucleotide sequence, homologue, variant or fragment thereof that is operably linked to the promoter whose promoter activity is being measured. The promoter activity may be measured directly by measuring the amount of RNA transcript produced, for example by Northern blot or indirectly by measuring the product coded for by the RNA transcript, such as when a reporter gene is linked to the promoter.

According to a further aspect of the invention, the above described polynucleotide sequences (and encoded proteins) can be used for the biosynthesis of (novel) triterpenoid sapogenin compounds. To illustrate this further, without the purpose of being limitative, one is refered to the below Example section.

The following examples are intended to promote a further understanding of the present invention. While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein. EXAMPLES

I. INTRODUCTION

EXAMPLE 1. Triterpenoid sapogenins in plant cultures.

In an attempt to detect triterpenoid sapogenins from plant cultures, we analyzed hairy roots of the model legume Medicago truncatula transformed with a Gateway plasmid pK7WG2D-GUS, containing a non-functional β-glucuronidase gene (GUS) expressed from a 35S promoter (Pollier et al., 2011). The hairy roots were grown for 3 weeks in 30 ml Murashige and Skoog basal salt mixture including vitamins (Duchefa) prior to an organic extraction and gas chromatography-mass spectrometry (GC-MS) analysis. The roots were harvested from the culture medium, frozen in liquid nitrogen and ground to a fine powder. A total metabolite extraction was performed on this ground material using 1 ml of methanol. The methanol phase was evaporated to dryness and the subsequent pellet was extracted with 1 ml hexane, since the triterpenoid sapogenins are extremely hydrophobic in nature. The hexane phase was separated from the undissolved residue, evaporated to dryness and trimethylsilylated as described (Radosevich et al. 1985). This derivatized material was subjected to GC-MS (GC model 6890, MS model 5973, Agilent) analysis where, a 1 μΙ aliquot was injected in splitless mode into a VF-5ms capillary column (Varian CP9013, Agilent) and operated at a constant helium flow of 1 ml/min. The injector temperature was set to 280 °C and the oven temperature was held at 80 °C for 1 min post injection, ramped to 280 °C at 20 °C/min, held at 280 °C for 45 min, ramped to 320 °C at 20 °C/min, held at 320 °C for 1 min, and finally cooled down to 80 °C at 50 °C/min at the end of the run. The MS transfer line was set to 250 °C, the MS ion source to 230 °C and the quadrupole to 150 °C, throughout.

Owing to the hydrophobicity of sterols, we detected multiple sterols and sterol intermediates in the GC chromatogram of this extract (Fig. 2). The identity of these compounds was confirmed using the MS electron ionization (El) pattern described in literature (data not shown). However, we only detected trace amounts of erythrodiol and no other triterpenoid sapogenins in this chromatogram (Fig. 2) emphasizing their low abundance in a glycosyl-free form in the hairy roots. Due to this inability to detect triterpene sapogenins from plant cultures we engineered a yeast strain capable of accumulating detectable amounts of these valuable compounds.

II. PRODUCTION OF TRITERPENOID SAPOGENINS IN MICROBIAL CULTURES

EXAMPLE 2. Generation of yeast strain TM1 with modified sterol biosynthesis. Triterpene saponin and sterol biosynthesis depend on the same precursor, i.e. oxidosqualene (Fig. 1). To enable maximal and a controllable flux towards the heterologous production of the desired triterpene compounds, we first engineered the endogenous sterol synthesis of the model yeast Saccharomyces cerevisiae, which leads to ergosterol as a major compound. The ergosterol biosynthetic pathway of the Saccharomyces cerevisiae strain S288c BY4742 was modified as described (Kirby et al., 2008) with adaptations. The lanosterol synthase gene (ERG7) (GenBank accession number N M_001179202) was made conditionally down-regulatable by replacing the native ERG7 promoter with a methionine-repressible MET3 promoter as described for ERG9 in S. cerevisiae CEN. PK 113-7D (Asadollahi et al., 2008), to generate strain TM1. The amount of ergosterol produced by TM1 in the presence of different concentrations of methionine was quantified using the sterol quantification method as described (Arthington-Skaggs et al., 1999). A 60 % reduction in ergosterol accumulation was observed in TM1 with 1.5 mM methionine when compared to wild type cells. Further, a truncated, feedback-uncoupled, copy of isoform 1 of the rate limiting enzyme 3- hydroxy-3-methylglutaryl-CoA reductase (tHMGl) (GenBank accession number NM_001182434) was generated as described (Polakowski et al., 1998). The tHMGl was cloned into the multiple cloning site (MCS) 1 of the high copy number plasmid pESC-U A (Agilent Technologies) behind the galactose inducible GAL10 promoter to generate pESC-\J RA[GAL10/tHMGl], which was transformed into TM1 to generate strain TM5.

EXAMPLE 3. Generation of 3-amyrin producing yeast strain TM3. The Glycyrrhiza glabra β-amyrin synthase (GgbAS) (GenBank accession number AB037203; (Hayashi et al., 2001)) was cloned into the MCS 2 of plasmid pESC-U RA[GAL10/tHMGl] to generate pESC- U RA[GAL10/tHMGl; GALl/bAS]. Strain TM1 was transformed with this plasmid using the lithium acetate mediated transformation method to generate strain TM3. To validate the production of β- amyrin in TM3, strains TM3 and TM5 were first precultured in Minimal synthetic defined (SD) base containing drop out (DO) supplement -Ura (SD -Ura) (Clontech) medium for 18-20 h at 30 °C and 250 rpm. The precultures were washed prior to inoculating Minimal SD Base Gal/Raf containing DO supplement -Ura (SD Gal/Raf -Ura) (Clontech) medium to a starting optical density of 0.25. The cultures were incubated as before for 24 h, prior to addition of 10 mM methionine to a final concentration of 1.5 mM, following which they were incubated further for 48 h. Yeast cells from a 1 ml culture were used for extraction and gas chromatography-mass spectrometry (GC-MS) analysis as described (Kirby et al., 2008) with modifications. The cell pellet was resuspended in an equal volume of 40 % potassium hydroxide and 50 % ethanol prior to lysis by boiling at 95 °C for 10 min. An organic extraction was performed on the lysate using an equivalent volume of hexane and vortexing at high speed for 1 min. The hexane extraction was repeated thrice before the phases were pooled and evaporated to dryness. A trimethylsilyl derivatization was performed on the dried material, and used for GC-MS analysis. The GC chromatograms showed the presence of a single peak at 27.2 min corresponding to 36.2 mg/L of β-amyrin in TM3 but not in TM5 (Fig. 3). The El pattern for this peak was identical to a standard of β-amyrin (Extrasynthese) (data not shown). However, when a similar extraction and GC-MS analysis was performed on 1 ml of spent medium without yeast cells no β- amyrin could be detected from either TM3 or TM5 (Fig. 3). In conclusion, we were able to engineer a yeast strain that is capable of synthesizing β-amyrin in significant amounts, however the β-amyrin that is generated is not secreted to the extracellular medium.

EXAMPLE 4. Generation of lupeol producing yeast strain TM6.

The Arabidopsis thaliana lupeol synthase (AtLUSl) (GenBank accession number U49919; (Herrera et al., 1998)) was cloned into the MCS 2 of plasmid pESC-URA[GAL10/tHMGl] to generate pESC- URA[GAL10/tHMGl; GALl/AtLUSl]. The plasmid was transformed into strain TM1 to generate a lupeol producing strain TM6. The production of lupeol by strain TM6 was verified by culturing as described in Example 3 and analyzing the trimethylsilylated fraction by GC-MS. A single peak at 28.9 min corresponding to 46.3 mg/L of lupeol with an El pattern identical to a standard of lupeol (Extrasynthese) (data not shown), was observed in the GC chromatograms of TM6 but not TM5. Again, no lupeol was detected in the spent medium of strains TM6 and TM5 (Fig. 4). It can thus similarly be concluded that, although lupeol is synthesized by the engineered yeast strain, it is not secreted to the growth medium.

EXAMPLE 5. Cvclodextrin facilitates secretion of β-amyrin and lupeol into the medium.

Strains TM3, TM6 and TM5 were cultured as in Example 2 with modifications. The precultures were prepared and inoculated into SD Gal/ af -Ura medium and incubated for 24 h as described. Along with methionine, 250 mM methyl^-cyclodextrin (CAVASOL©, Wacker Quimica Iberica S.A.) was also added to a final concentration of 5 mM and the cultures were incubated further under the same conditions. Post 24 h incubation, 250 mM methyl^-cyclodextrin was added once again to a concentration of 5 mM and the cultures were incubated further for 24 h. The cells and spent medium were harvested from 1 ml of the culture and processed separately for extraction and GC-MS analysis as described in Example 3. GC chromatograms confirmed the presence of β-amyrin and lupeol in the cell pellet as well as in spent medium of strains TM3 and TM6, respectively but not TM5, with higher concentrations of both β-amyin and lupeol quantified from the extracts of spent medium (37.3 mg/L and 164.2 mg/L, respectively) when compared to cell pellet (20.4 mg/L and 41.7 mg/L, respectively) (Fig. 5). Additionally, the total concentration of both β-amyrin and lupeol is found to be 1.6 fold and 4.4 fold, respectively, higher in cultures treated with cyclodextrin when compared to non-treated controls. The absence of β-amyrin and lupeol in the extracts obtained from spent medium of cells cultured in the absence of cyclodextrin and vice versa strongly underscore the effects of cyclodextrins on the secretion of triterpene sapogenin backbones into the medium.

Further we determined if cyclodextrin facilitates the secretion of triterpene sapogenin backbones in a dose dependent manner. For this we employed the β-amyrin producing strain TM3 and applied cyclodextrin at different times during culturing. A total of 7 culturing conditions were set up which included, an untreated control (C) and 6 treated samples, with cyclodextrin added to a concentration of 5 mM each time. To samples II, 12 and 13 cyclodextrin was added on Day 1 immediately after inoculation into SD Gal/Raf -Ura medium. To samples 12 and 13 an additional dose of cyclodextrin was added on Day 2 together with addition of methionine. Additionally, to sample 13 a third dose of cyclodextrin was added on Day 3. Further, to samples Rl, R2, and AR1 cyclodextrin was added on; Day 2 only, Day 2 and Day 3, and Day 3 only, respectively. Extractions were performed on the spent medium of all the samples on Day 4 and quantified for β-amyrin using GC-MS as described. Surprisingly, we observed a direct corelation between the amount of β-amyrin quantified from the spent medium and the number of times cyclodextrin was added to the sample, thereby suggesting the dose dependent nature of this secretion (Fig. 6). For the purpose of the following experiments, being the generation of sapogenins derived from its precursors (e.g. β-amyrin, lupeol), we decided to employ condition R2 for all our subsequent experiments. In this way, the excessive secretion from the cells into the medium and hence loss of β-amyrin, which is the precursor for the consecutive cytochrome P450 monooxygenases (CYPs), is prevented.

EXAMPLE 6. Secretion of triterpene sapogenins from strains TM10, TM11, TM12 and TM22. To produce triterpene sapogenins in our yeast strain we modified β-amyrin and lupeol with three characterized cytochrome P450 monooxygenases (CYPs), CYP716A12 (GenBank accession number FN995113; (Carelli et al., 2011)), CYP88D6 (GenBank accession number AB433179; (Seki et al., 2008)) and CYP93E2 (GenBank accession number DQ335790; (Li et al., 2007)). The CYPs need a CYP reductase (CPR) as a redox partner for their activity. Therefore, we simultaneously cloned the A. thaliana CPR, ATRl (At4g24520) along with the CYPs. Both the CYPs and CPR were PCR amplified and cloned into the entry vector pDONR221 by Gateway™ recombination (InVitrogen Life Technologies). Further the CYPs were Gateway recombined into the high copy number expression vector, pAG423GAL-ccdB (Addgene plasmid 14149) containing the GAL1 promoter and HIS3 auxotrophic marker to generate the plasmids pAG423[GALl/CYP716A12], pAG423[GALl/CYP88D6] and pAG423[GALl/CYP93E2]. The CP was Gateway recombined into the high copy number expression vector pAG425GAL-ccdB (Addgene plasmid 14153) having the GAL1 promoter and LEU2 auxotrophic marker to generate plasmid pAG425[GALl/AtATRl].

We transformed strain TM3 with the plasmid pAG425[G ALl/AtATRl] in combination with either pAG423[GALl/CYP716A12], pAG423[GALl/CYP88D6], pAG423[GALl/CYP93E2] or pAG423GAL-ccdB to generate strains TM10, TM11, TM12 and TM26, respectively. The spent medium of strains TM10, TM11, TM12 and TM26, cultured in SD Gal/Raf -Ura/-His/-Leu medium with cyclodextrin treatment as described in Example 5, was analyzed by GC-MS. Peaks corresponding to erythrodiol, oleanolic aldehyde and oleanolic acid at high concentration were detected in the GC chromatograms of extract from TM10 indicating the C-28 hydroxylation of β-amyrin mediated by CYP716A12. Similarly, 11- hydroxy- -amyrin and ΙΙ-οχο-β-amyrin were detected in the chromatogram of strain TM11 and 24- hydroxy- -amyrin in the extract of strain TM12. The identity of all the hydroxylated β-amyrin peaks was confirmed by comparing their El patterns against, available standards (erythrodiol, oleanolic acid (Extrasynthese)), or previous reports when a commercial standard was unavailable. No hydroxylated β- amyrin was detected in the chromatogram of TM26 (Fig. 7). The presence of triterpene sapogenins in the growth medium of strains TM10, TM11 and TM12 suggest the role of cyclodextrin in the secretion of oleanane type sapogenins from the yeast cells into the culture medium. Next we determined if cyclodextrin could also facilitate the secretion of lupane type triterpene sapogenins from the cells to the culture medium. For this we generated strains TM22 and TM28 by transforming strain TM6 with the plasmids pAG425[G ALl/AtATRl] and pAG423[GALl/CYP716A12] or pAG423GAL-ccdB, respectively. Strains TM22 and TM28 were cultured as described using SD Gal/Raf - Ura/-His/-Leu medium with cyclodextrin treatment and the spent medium was used for extraction and GC-MS analysis. We detected 7.2 mg/L betulin and 2.4 mg/L betulinic acid in the chromatograms of TM22 but not TM28 indicating the C-28 hydroxylation of lupeol by CYP716A12 (Fig. 8). The detection of hydroxylated and carboxylated lupeol in the growth medium further supports the effect of cyclodextrin in the secretion of lupane type sapogenins as well.

I I I. PRODUCTION OF TRITERPENOI D SAPOGENI NS I N PLANT CU LTU RES

EXAMPLE 7. Cyclodextrin facilitates secretion of triterpene sapogenins from Medicaqo truncatula. We then determined if cyclodextrins could also facilitate the production and/or secretion of triterpene sapogenins from the model legume M. truncatula by analyzing the spent medium of M. truncatula hairy roots transformed with pK7WG2D-GUS. The hairy roots were grown for 2 weeks in 20 ml Murashige and Skoog basal salt mixture including vitamins (Duchefa) prior to addition of 250 mM cyclodextrin to a final concentration of 25 mM in combination with or without 100 μΜ methyl jasmonate and compared to, untreated and 100 μΜ methyl jasmonate only treated controls. The roots were harvested 48 h after treatment and the spent medium was extracted and analyzed by GC-MS. Surprisingly, the triterpene sapogenins erythrodiol, oleanolic acid and oleanolic aldehyde, corresponding to the building blocks of the most abundant saponins produced by the hairy roots (Pollier et al., 2011) could now be detected in the GC chromatograms of medium of roots treated with cyclodextrin, but not in control roots (Fig. 9). We compared El patterns against available standards to confirm the identity of erythrodiol and oleanolic acid (Extrasynthese), and previous reports for oleanolic aldehyde. Additionally, a significant increase, of upto 150 fold for erythrodiol and 2 fold for oleanolic acid, was noted when cyclodextrin was combined with methyl jasmonate treatment (Fig. 9). This confirms the role of cyclodextrins in the secretion of scarcely intracellularly accumulating saponin intermediates, the sapogenins, from plant cultures into the culturing medium, as described in Example 1.

IV. IDENTIFICATION AND CHARACTERIZATION OF NOVEL SAPONIN BIOSYNTHETIC GENES

EXAMPLE 8. Transcript profiling of MeJA-treated Bupleurum falcatum reveals a novel plant cytochrome P450

The genus Bupleurum constitutes of perennial herbs and forms an integral part of Asian traditional medicine in which it is used either alone, or in combination with other ingredients for the treatment of common colds, fever and inflammatory disorders in the form of over the counter herbal teas. Saikosaponins constitute the largest class of secondary metabolites in Bupleurum and account for ~7 % of the total dry weight of roots. More than 120 closely related glycosylated oleanane- and ursane-type saikosaponins have been identified from this genus that can be distinguished only by the positions and numbers of, double bonds in rings C and D and oxygenation patterns on C-16, C-23, C-28 and C-30 (Fig. 10) (Ashour and Wink, 2011). The presence of oxygenations at various positions on saikosapogenins suggests the presence of specific enzymes, generally CytP450s, capable of catalyzing these modifications on the β-amyrin and/or a-amyrin backbone in the genus Bupleurum. However, to date not a single CytP450 or oxido-reductase involved in triterpene sapogenin biosynthesis has been identified from Bupleurum species. To identify new saponin biosynthesis genes, we performed a genome-wide cDNA-AFLP based transcript profiling on the roots of hydroponically grown B. falcatum plants. B. falcatum seeds, obtained from a commercial source (www.SandMountainHerbs.com), were sown in soil, and 2 weeks after germination, seedlings were transferred to aerated hydroponics medium containing l g/L 10-30-20 salts (Scotts, Ohio, USA), pH 6.5. Plants were grown at 16 h/8 h light/dark regime, at 21°C. The pH was monitored daily and adjusted to 6.5, by adding KOH to the hydroponics medium. Three weeks after the plants were transferred to the hydroponics medium, they were treated with 50 μΜ methyl jasmonate (MeJA) (dissolved in ethanol (EtOH)) or an equivalent amount of EtOH as a control, by adding the EtOH or MeJA solution directly to the hydroponics medium. For transcript profiling, roots were harvested 0, 0.5, 1, 2, 4, 8 and 24 h after treatment, frozen in liquid nitrogen, and stored at -70°C. For each sample, 3 individual plants were pooled.

A full genome-wide cDNA-AFLP based transcript profiling on the roots of hydroponically grown B. falcatum plants was carried out as described in Vuylsteke et al. (2007). Gel images were analyzed with the AFLP-QUANTA P O software (Keygene, Wageningen, The Netherlands), allowing accurate quantification of band intensities. Extraction and analysis of expression data of all individual bands, selection of tags displaying differential expression, cluster analysis, sequencing, and BLAST analysis was performed as described (Rischer et al., 2006).

Using the complete set of 128 BstY\+l/Mse\+2 primer combinations, the expression of a total of 18,800 transcript tags was monitored over time. In total, 1,771 MeJA-responsive transcript tags were isolated (hereafter referred to as BF tags). Direct sequencing of the reamplified BF tags gave good-quality sequences for 1217 (68.7 %) of the fragments. To the remaining 554 tags (31.3 %), no unique sequence could be attributed unambiguously, indicating that they might not represent unique gene tags, hence, these tags were not considered for further analysis. A blast search with the nucleotide sequences of the 1217 unique cDNA-AFLP tags led to the annotation of 776 (63.7 %) of the BF tags. Average linkage hierarchical clustering analysis of the expression profiles of the 776 annotated BF tags showed that, upon MeJA treatment the selected genes are either transcriptionally activated or transcriptionally repressed. The activated gene tags can be divided into different subclusters, based on the timing of the MeJA-response. In one subcluster, genes are activated within 2 h after the MeJA treatment, and their expression remains high thereafter. In this group, tags corresponding to genes encoding enzymes that catalyze early steps in the triterpene saponin biosynthesis, including squalene synthase (SQS) and β-amyrin synthase (MS) can be found. These tags displayed an almost identical expression pattern, suggesting a tight co-regulation, and reached maximum levels of expression 8-24 h post-elicitation (Fig. 11). The gene tag BF567 (hereafter named CYP716A021) is tightly co-regulated with these genes (Fig. 11), and shows homology to the M. truncatula gene encoding the cytochrome P450 enzyme CYP716A12 that was recently shown to oxidize β-amyrin in a sequential three-step oxidation on C-28 to yield oleanolic acid through erythrodiol (Carelli et al., 2011; Fukushima et al., 2011). The full-length open reading frame (BF567, hereafter named CYP716A021) corresponding to the gene tag CYP716A021 was picked up from a B. falcatum Uncut Nanoquantity cDNA library (Pollier et al., 2011b). Using the primers (sense, 5'-CCTCCTTATACATTCGTTCCATTC-3' (SEQ ID NO: 20) and antisense, 5' -TT AG G GTCTACTTTCTCCC ATTTG -3' (SEQ ID NO: 21), the full-length coding sequence of CYP716A021 (SEQ ID NO: 1) corresponding to the gene tag CYP716A021 was picked up from the screening of a B. falcatum Uncut Nanoquantity cDNA library (custom-made by Invitrogen, Carlsbad, CA, USA) as reported (Pollier et al., 2011b). The full-length open reading frame (FL-O F) of CYP716A021 was PCR amplified for Gateway™ cloning into the entry vector pDONR221 using the primer pair P19 ( G G G G AC AAGTTTGT AC A AA AA AG C AG G CTTA ATG G AACTTTCTATC ACT) + P20

(GGGGACCACTTTGTACAAGAAAGCTGGGTATTAAGATGGAGATTTGTG). The entry clone of CYP716A021 was recombined into the high copy number expression vector pAG423GAL-ccdB (Addgene plasmid 14149) with the GAL1 promoter and HIS3 auxotrophic marker, resulting in pAG423[GALl/CYP716A021].

EXAMPLE 9. In vivo activity of CYP716AQ21 in yeast strain TM7.

Yeast strain TM7 was generated by super-transforming strain TM3 (see Example 3) with plasmids pAG423[GALl/CYP716A021] and pAG425[GALl/AtATRl], expressing CYP716A021 and the A. thaliana CytP450 reductase (CPR), AtATRl (At4g24520), respectively from the galactose inducible GAL1 promoter. In parallel, a control strain TM26 harboring only pAG425[G/AZJ/ 'AtATRl] in TM3 was also generated. Cell pellets analyzed by GC-MS showed the presence of a unique new peak eluting at 31.8 min in TM7 (Fig. 12A), but not TM26 (Fig. 12C). The El pattern of this peak corresponded to a hydroxylated derivative of β-amyrin, with the alcohol function on either the D or E ring of β-amyrin (Fig. 12D).

Since CYP716A021 was tentatively annotated as a homolog of the M. truncatula CYP716A12 (GenBank accession number FN995113; (Carelli et al., 2011)), we compared the GC elution time and El pattern of the new peak in TM7 with a standard of erythrodiol (28-hydroxy- -amyrin). We also generated strain TM10 by transforming plasmid pAG423[GALl/CYP716A12] along with pAG425[GALl/AtATRl] in TM3, and compared its GC-MS profile with TM7, TM26 and an erythrodiol standard. A peak corresponding to the elution time and El of standard erythrodiol (Fig. 12E) was observed at 32.5 min in the GC chromatogram of TM10 (Fig. 12B) but not TM7 and TM26, indicating that CYP716A021 hydroxylates β- amyrin at a position different than CYP716A12. Therefore, we looked at the oleanane-type triterpenoid sapogenins found in Bupleurum (Ashour and Wink, 2011) and narrowed down on the possible hydroxylation positions of CYP716A021 on rings D and E of β-amyrin to C-16, C-21 and C-29 (Fig. 10), which are positions for which a CytP450 has not been characterized to date.

EXAMPLE 10. Effect of CPR:CytP450 ratio on in vivo activity of CYP716AQ21.

The endoplasmic reticulum (ER) localized CPRs are flavoproteins, containing both a redox cofactor flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), that serve as electron donor proteins for several ER oxygenases, including CytP450s. Therefore, optimal interaction between CPR and CytP450 is essential to allow the reducing equivalents from NADPH to pass from the CPR to the CytP450 (Reed and Backes, 2012). In an attempt to increase the efficiency of hydroxylation of β-amyrin by CYP716A021, we determined the effect of the ratio of CPR to CytP450, by varying the expression level of AtATRl while keeping the expression of CYP716A021 constant.

The CPR:CytP450 ratios between 1:5 and 1:30 have been reported to be ideal for the efficient functioning of yeast and mammalian CytP450s (Reed and Backes, 2012), therefore we expressed the AtATRl from either an integrated (pAG305, 1 copy per cell), low-copy number (pAG415, 3-5 copies per cell), or high-copy number (pAG425, 10-40 copies per cell) vector, in combination with CYP716A021 always expressed from the high-copy number plasmid (pAG423, 10-40 copies per cell). Thus, we generated two strains TM8 and TM9, overexpressing pAG423[GALl/CYP716A021] along with pAG305[GALl/AtATRl] or pAG415[GyAZJ 4tA77?.i], respectively and compared the amount of hydroxylated β-amyrin produced by these strains with that of TM7. In accordance with this assumption, strain TM9, accumulated higher levels of hydroxylated β-amyrin compared to TM8 and TM7, with the lowest accumulation in the strain expressing the integrated copy of CPR (Fig. 13A). Therefore, we expressed AtATRl from the low-copy number vector pAG415, for further experiments.

EXAMPLE 11. Secretion of triterpene sapogenins from strains TM9

For the following experiments with strain TM9, we employed condition R2 to avoid the excessive secretion and hence loss of β-amyrin, the precursor for CYP716A021, from the cells into the medium. Surprisingly, the hydroxylated β-amyrin eluting at 31.8 min was only observed in the GC chromatograms of the spent medium and not cell pellets of TM9 upon MβCD treatment (Fig. l3B), suggesting the complete secretion of the hydroxylated product from the yeast cells into the medium. We also determined the specificity of the type of CD used, on the sequestering of hydroxylated β- amyrin from the cells into the spent medium of strain TM9. The most abundant variants of CD are α, β and yCD which have 6, 7 and 8 glucose units, respectively. Therefore, we applied aCD, CD, yCD, Random M CD (RM CD) or M CD to a final concentration of 5 mM as in condition R2 and analyzed the spent medium on Day 4, for quantification of the amount of hydroxylated β-amyrin secreted into the medium. Sequestering was only observed with the CD and its methylated versions and the highest amount of hydroxylated β-amyrin was detected upon RM CD and M CD treatment, suggesting a strong specificity of the methylated forms of CD over the unmethylated forms, for sequestering of triterpenoid sapogenins from yeast cells (Fig. 13C). EXAMPLE 12. Transcript profiling of MeJA-treated Maesa lanceolate/ reveals a novel plant cytochrome P450

Maesa lanceolata, a member of the Myrsinaceae family, is a shrub or small tree indigenous to Africa. African traditional healers use extracts and/or parts of the plant for the treatment of a wide range of diseases including infectious hepatitis, bacillary dysentery, impetigo, ozena, dermatoses and neuropathies. Methanol extracts of M. lanceolata leaves are rich in maesasaponins and have been shown to possess virucidal, molluscicidal, fungistatic and antimutagenic activities (Sindambiwe et al., 1998). The maesasaponins identified so far are derived from an oleanane skeleton via modifications of the β-amyrin backbone, resulting in a characteristic C-13,28 hemiacetal or ester bridge and oxidations on C-16, C-21 and C-22 (Fig. 14) (Foubert et al., 2010; Manguro et al., 2011). The hemiacetal or ester bridge between C-13 and C-28 is thought to occur through the reaction between a C-13 hydroxyl and C-28 aldehyde or carboxyl group, respectively (Vincken et al., 2007). The presence of these diverse oleanane maesasaponins suggests the presence of a β-amyrin specific OSC (or β-amyrin synthase) along with specific CytP450s catalyzing oxygenations at C-16, C-21, C-22 and C-28 in M. lanceolata. However, to date not a single triterpene saponin biosynthetic gene has been identified from Maesa. To identify new saponin biosynthetic genes, we performed a transcript profiling on methyl jasmonate (MeJA) treated M. lanceolata axenic shoot cultures. M. lanceolata axenic shoot cultures were generated and maintained as described (Faizal et al., 2011). For elicitation, each pot of shoot culture was sprayed with 2 ml deionized water containing 0.05% (v/v) Tween-20 in combination with 500 μΜ MeJA (10 μΙ of 100 mM stock dissolved in ethanol) or an equivalent amount of ethanol as control. For transcript profiling, samples were collected 0, 0.5, 1, 2, 4, 8, 24 and 48 h after elicitor or mock treatments. For each sample, 3 different plants were pooled. Using the complete set of 128 BstY\+l/Mse\+2 primer combinations, a genome-wide cDNA-AFLP transcript profiling analysis (Vuylsteke et al., 2007; see also Example 8) was carried out to monitor the expression of a total of 13,558 transcript tags over time. In total, 733 MeJA-responsive transcript tags were isolated (hereafter referred to as ML tags). Direct sequencing of the reamplified ML tags gave good quality sequences for 545 (74.4%) of the fragments. To the remaining 188 tags (25.6%), no unique sequence could be attributed unambiguously, indicating that they might not represent unique gene tags and hence, these were not considered for further analysis. A blast search with the nucleotide sequences of the 545 unique cDNA-AFLP tags led to the annotation of 312 (57.2%) of the ML tags. Average linkage hierarchical clustering analysis of the expression profiles of the ML tags showed that, upon MeJA treatment the genes are either transcriptionally activated or transcriptionally repressed. The activated gene tags can be divided into different subclusters, based on their MeJA response time. In one subcluster, a gene tag that reached maximum levels of expression 24-48 h post-elicitation and corresponding to squalene epoxidase (SQE) can be found (Fig. 15). The gene tags ML257 and ML593, corresponding to CytP450s are tightly co-regulated with this gene (Fig. 15). The gene tag ML257 shows homology to the M. truncatula CYP716A12 that was shown to oxidize β-amyrin in a sequential three- step oxidation on C-28 to yield oleanolic acid through erythrodiol (Carelli et al., 2011; Fukushima et al., 2011), and the gene tag ML593 shows homology to the A. thaliana steroid 22a-hydroxylase gene encoding a CytP450 enzyme that catalyzes the oxidation of sterols on the C-22 position (Fujita et al., 2006). The full-length open reading frame ML593 corresponding to the respective gene tag was picked up from a M. lanceolate/ Uncut Nanoquantity cDNA library (Pollier et al., 2011), using the primer pairs P27 (CTCTTGCATTCAATCCGAAAC) + P28 (AGCAAAGAATGCCTTGGCTA). The FL-O F of ML593 was PCR amplified for GatewayTM cloning in pDONR221 using the primer pairs P39 ( G G G G AC AAGTTTGTAC A AA A AAG C AG G CTT AATGTG G GTAGTGG G ATTA) + P40

( G G G G ACC ACTTTGTAC AAG A AAG CTG G GTATC ACTTGTTTTTCTTG GT) . The entry clone ML593 was GatewayTM recombined into the high-copy number expression vector pAG423GAL-ccdB behind the galactose inducible GAL1 promoter and having the HIS3 auxotrophic marker, resulting in pAG423[GAi.1/ML593].

EXAMPLE 13. In vivo activity of ML593 in yeast strain TM21

To characterize the putative CytP450, ML593 from our transcript profiling, we generated strain TM21 from the β-amyrin producing strain TM3 (Table 4; see Example 3), by supertransforming with the plasmids pAG415[GAZJ 4tA77?:-] and pAG423[GAZJ//Wi.593]. The strains TM21 and TM27 (Table 4) were cultured in the presence of MfiCD and the spent medium analyzed by GC-MS. We observed a new peak eluting at 31.8 min corresponding to a hydroxylated β-amyrin in strain TM21 (Fig. 16A), but not in the control strain TM27 (Fig. 16C). The El pattern of this peak (Fig. 16A) corresponded to a hydroxylation on the D or E ring of the oleanane structure and was similar to that observed with strain TM9 (Table 4) expressing CYP716A021 (Fig. 16B). The strong similarity between the elution time and El pattern of the hydroxylated β-amyrin in strain TM21 and TM9 further supports this assumption. We also observed that strain TM21 produced 8-fold more hydroxy β-amyrin than strain TM9, highlighting the better efficiency of ML593 for hydroxylating β-amyrin as compared to CYP716A021.

V. COMBINATORIAL BIOSYNTHESIS OF T ITE PENOID SAPOGENINS IN YEAST

EXAMPLE 14. Combinatorial biosynthesis using CYP716AQ21 and CYP716A12

Combinatorial biosynthesis also known as combinatorial biochemistry involves the combination of genes from different organisms, in a heterologous host to produce bioactive compounds by establishing novel enzyme-substrate combinations in vivo, which in turn could lead to the biosynthesis of novel natural products (Pollier et al., 2011). Although CYP716A021 is tentatively annotated as a homolog of CYP716A12, the GC elution time and El pattern of the β-amyrin hydroxylation product of CYP716A021 is different from erythrodiol (Fig. 12). We reasoned that if the two enzymes hydroxylate β-amyrin at two different carbon positions, it should be possible to combine the enzymes in the yeast strain TM3 and produce a combinatorial compound not produced by either of the enzymes alone. Therefore, we generated strain TM30 from TM3 by overexpressing the plasmids pAG415[GALl/AtATRl] and pAG423[GALl/CYP716A021-T2A-CYP716A12], where CYP716A021 and CYP716A12 are stitched together into a self-processing polyprotein via the 2A oligopeptide (de Felipe et al., 2006) which is expressed from a single galactose inducible GALl promoter. The self-processing polyprotein of CYP716A021 and CYP716A12, was generated by amplifying the FL-ORF of CYP716A021 without a stop codon and having a 3'- overhang of the partial T2A sequence using the primer pair P19 ( G G G G AC AAGTTTGTAC A AA AA AG C AG G CTTA ATG G AACTTTCTATC ACT) + P23

(ACCGCAUGTTAGCAGACTTCCTCTGCCCTCAGATGGAGATTTGTGGGGAT). The FL-ORF of CYP716A12 was amplified with a 5'- overhang of the partial T2A sequence using the primers P24 (ATGCGGUGACGTCGAGGAGAATCCTGGCCCAATGGAGCCTAATTTCTATC) + P22

( G G G G ACC ACTTTGTAC A AG AA AG CTG G GTATTA AG CTTTGTGTG G ATA AAG G CG ) such that there was an overlap of 7 bp between the two amplified sequences. Since the primers P23 and P24 contain an Uracil each, the CYP716A021 and CYP716A12 were PCR amplified using the Pfu Turbo Cx polymerase (Stratagene). The purified gel fragments were used for Uracil-Specific Excision or USER™ Cloning (New England Biolabs) to generate two fragments with complementary sticky ends which were ligated in vitro using the T4 DNA ligase (Invitrogen). The ligated DNA product was once again gel purified and used as template for amplification with the primers P19 + P22. This amplicon was Gateway™ recombined into pDON 221, sequence verified and further recombined into pAG423GAL-ccdB to generate pAG423[GALl/CYP716A021-T2A-CYP716A12].

The spent medium of strain TM30 cultured in the presence of M CD was analyzed by GC-MS and compared to the GC chromatograms of spent medium from strains TM9, and TM17 overexpressing pAG423[GALl/CYP716A12] and pAG415[GALl/AtATRl] in TM3. We observed a unique peak at 40.5 min in strain TM30 (Fig. 17A) but not TM9 (Fig. 17B) and TM17 (Fig. 17C), strongly supporting the fact that CYP716A021 and CYP716A12 catalyze hydroxylations of two different carbons on β-amyrin. Additionally, the El pattern of this peak suggested the presence of carboxyl and alcohol functions on β- amyrin, indicating the C-28 carboxylation by CYP716A12 and the C-16, C-21 or C-29 hydroxylation by CYP716A021. Considering the close proximity of C-16 and C-28 on the β-amyrin molecule (Fig. 10) and the tentative annotation of CYP716A021 as a homolog of CYP716A12 we reasoned that CYP716A021 might hydroxylate C-16 of β-amyrin. Therefore, we compared the GC chromatogram and El pattern of an echinocystic acid standard (Fig. 17E) with that of the new peak at 40.5 min in TM30 (Fig. 17D). However, the GC elution time and fragmentation of the new peak at 40.5 min did not match that of echinocystic acid ^,16a-dihydroxyolean-28-oic acid) ruling out the possibility of a C-16 a- hydroxylation by CYP716A021. Due to the absence of authentic 3β,16β^ϋ^ΓθχγοΙθ3η-28-ο acid, 3β,21β^ϊΙ^ΓθχγοΙθ3η-28-ο^ acid and 3β,29α^ϋ^ΓθχγοΙθ3η-28-ο acid standards we could not confirm the identity of the combinatorial compound. In conclusion, using a combinatorial approach in S. cerevisiae we revealed that CYP716A021 is not a C- 28 oxygenase but, a novel cytochrome P450 that encodes a β-amyrin

involved in the synthesis of saikosaponins in Bupleurum.

EXAMPLE 15. Combinatorial biosynthesis using ML593 and CYP716A12

First, to confirm the identity of ML593 as a functional homolog of CYP716A021, we generated strain TM31, similar to strain TM30 (Table 4) (see Example 14). Strain TM31 was created by supertransforming TM3 with the plasmids pAG415[G,AZJ 4t.A77?:-] and pAG423[GALl/ML593-T2A-

CYP716A12], where ML593 and CYP716A12 form a selfprocessing polyprotein stitched together by the

2A oligopeptide (de Felipe et al., 2006) and is expressed from a single GAL1 promoter. The strains

TM31, TM17 and TM21 (Table 4) were cultured in the presence of I ^CD and the spent medium was analyzed using GC-MS. Similar to strain TM30 (Fig. 18D), we observed a new peak eluting at 40.5 min in strain TM31 (Fig. 18a), but not the strains TM17 (Fig. 18B) and TM21 (Fig. 17C) expressing only the

CytP450 CYP716A12 and ML593, respectively. From the El pattern of this peak we could confirm its identity as being the same as in strain TM30, which together with the lack of homology with echinocystic acid suggests a β-hydroxylation on C-21, the only remaining position commonly hydroxylated between CYP716A021 and ML593. Although the CYP716A021 and ML593 belong to different CytP450 subfamilies (CYP716A and CYP90B, respectively), in our yeast strain they demonstrate the same catalytic activity.

EXAMPLE 16. Combinatorial biosynthesis using ML593 and CYP88D6

To generate a combinatorial scaffold using β-amyrin we expressed ML593 (see Examples 12-13) and CYP88D6 in strain TM3. The CytP450 CYP88D6 (GenBank accession number AB433179; (Seki et al., 2008)) catalyzes a two-step oxidation of β-amyrin to ΙΙ-οχο-β-amyrin through a ll-hydroxy^-amyrin intermediate. First, we confirmed the activity of CYP88D6 in our β-amyrin producing strain by generating strain TM18 (Table 4) and culturing it in the presence of I ^CD along with the control strain TM27. Therefore, the full-length open reading frame (FL-ORF) of CYP88D6 was PCR amplified using the primers P45 ( G G G G AC A AGTTTGT AC A AA A AAG C AG G CTTA ATG G A AGT AC ATTG G GTTTG ) + P46 (GGGGACCACTTTGTACAAGAAAGCTGGGTACTAAGCACATGAAACCTTTA) and cloned into pDONR221. The CytP450 CYP88D6 was then Gateway™ recombined into the high-copy number expression vector pAG423GAL-ccdB (Addgene plasmid 14149). GC chromatograms of the spent medium of strain TM18 showed the presence of four unique peaks (Fig. 19c) that were absent in the control strain TM27 (Fig. 19d). Two of these peaks eluting at 25.4 min and 37.5 min corresponded to 11-hydroxy β-amyrin and 11-oxo β-amyrin (Seki et al., 2008), respectively. The two remaining peaks eluting at 24.4 min and 26.6 min could not be assigned an identity despite their clear El pattern. The highest mass observed in the mass spectra extracted from these peaks (Fig. 19e) was lower than that of trimethylsilylated β-amyrin (M+=498), indicating their possibly non-triterpenoid origin. These additional peaks observed in our yeast strain were not reported when the CYP88D6 was expressed in a wild type yeast strain expressing a β-amyrin synthase from Lotus japonicus (Seki et al., 2008). Further, we generated strain TM32 by supertransforming strain TM3 with the plasmids pAG415[GAZJ 4tA77?-!] and pAG423[GALl/ML593-T2A-CYP88D6], where ML593 and CYP88D6 are stitched together with the 2A oligopeptide resulting in the generation of a self-processing polypeptide (de Felipe et al., 2006). The self-processing polyprotein of ML593 and CYP88D6, was generated by amplifying the FL-ORF of ML593 without a stop codon and having a 3'- overhang of the partial T2A sequence using the primer pair P39

( G G G G AC AAGTTTGTAC AA A AA AG C AG G CTTA ATGTG G GTAGTG G G ATTA) + P47

(ACCGCAUGTTAGCAGACTTCCTCTGCCCTCCTTGTTTTTCTTGGTGACCT). The FL-ORF of CYP88D6 was amplified with a 5'- overhang of the partial T2A sequence using the primers P48 (ATGCGGUGACGTCGAGGAGAATCCTGGCCCAATGGAAGTACATTGGGTTT) + P46

(GGGGACCACTTTGTACAAGAAAGCTGGGTACTAAGCACATGAAACCTTTA) such that there was an overlap of 7 bp between the two amplified sequences. Since the primers P48 and P47 contain an Uracil each, the ML593 and CYP88D6 were PCR amplified using the Pfu Turbo Cx polymerase (Stratagene). The purified gel fragments were used for USER™ Cloning (New England Biolabs) to generate two fragments with complementary sticky ends which were ligated in vitro using the T4 DNA ligase (Invitrogen). The ligated DNA product was used as template for amplification with the primers P39 + P46. This amplicon was Gateway™ recombined into pDONR221, sequence verified and further recombined into pAG423GAL-ccdB to generate pAG423[GALl/ML593-T2A-CYP88D6]. The spent medium of strain TM32 (Fig. 19a) cultured in the presence of M CD was compared with that of strains TM21 (Fig. lb), TM18 (Fig. 19c) and TM27 (Fig. 19d). Strain TM32 showed the presence of two unique peaks eluting at 32.1 min and 44.7 min that could correspond to lla,21 (?)-dihydroxy β-amyrin and ll-oxo-21 (?)-hydroxy β-amyrin based on their El pattern, respectively. EXAMPLE 17. Combinatorial biosynthesis using ML593 and CaDDS

In an attempt to generate a dammarenediol producing yeast strain we expressed the plasmid pESC- URA[GAL10/tHMGl; GALl/CaDDS], harboring a dammarenediol synthase gene (CaDDS) from Centella asiatica (GenBank accession number AY520818; (Kim et al., 2009)), in the sterol modified yeast strain TM1, to generate strain TM33. The CaDDS was amplified with Xhol and Nhel containing primers P43 (GGGGACAAGTTTGTACAAAAAAGCAGGCTTActcgagATGTGGAAGCTGAAGATAGCA) + P44

(GGGGACCACTTTGTACAAGAAAGCTGGGTTgctagcTCAATTGGAGAGCCACAAGC G) to generate pESC- URA[GAL10/tHMGl; GALl/CaDDS].

We cultured strain TM33 and the control strain TM5 in medium containing M CD and analyzed the GC chromatograms obtained from the spent medium of both the strains. Unexpectedly, we found 3 new peaks in strain TM33 eluting at 27.2 min, 28.6 min and 33.5 min (Fig. 20a) that were absent in the control strain TM5 (Fig. 20b). We could confirm the identity of the peak eluting at 27.2 min as β-amyrin (Fig. 20c) based on its elution time and its El pattern. The peak at 28.6 min had a similar El pattern as β- amyrin and was confirmed as a-amyrin by comparing to a standard (Fig. 20d). The El pattern of the peak at 33.5 min could be interpreted to dammarenediol-ll (Spencer, 1981), but was not confirmed due to the lack of an authentic standard. Although, the CaDDS was initially reported as a putative bAS (Kim et al., 2005) and later characterized as a dammarenediol synthase (Kim et al., 2009), in our yeast strain the gene was capable of cyclizing 2,3-oxidosqualene to both β-amyrin and dammarenediol in addition to a third product, a-amyrin. In our yeast strain the relative amounts of a-amyrin, β-amyrin and dammarenediol-ll were in the ratio of 8.8:1.1:0.1, highlighting the very low dammarene synthase activity of CaDDS as opposed to its current characterization.

Strain TM37 was generated from strain TM33 by supertransforming with the plasmids pAG415[GALl/AtATRl] and pAG423[GALl/ML593] (see Table 4). The ML593 was characterized as a putative C-21 hydroxylase of β-amyrin (see Examples 12-13) and we determined the substrate specificity of this CytP450 by expressing it together with the multifunctional cyclase CaDDS. We cultured strain TM37 and the control strain TM38 in the presence of M CD and compared GC chromatograms for the presence of unique peaks. We identified two peaks eluting at 31.8 min and 33.2 min in the spent medium of TM37 (Fig. 21a), but not the control strain TM38 (Fig. 21b). The El pattern of both these peaks were identical (Fig. 21a) and the peak at 31.8 min corresponded to (most likely) 21-hydroxy β-amyrin (see Examples 12-13). Therefore, the second peak at 33.2 min could correspond to 21-hydroxy a-amyrin, but was not confirmed due to the absence of an authentic standard.

Table 4. List of yeast strains generated and used in this study.

Name Construct

S288c BY4742 MATa; his3Al; leu2A0; ura3A0; lys2A0

TM1 S288c BY4742; P _erg7 ::P _MET3 -ERG7

TM3 TM1; pESC-URA[GAL10/tHMGl; GALl/GgbAS] (36.2 mg/L β-amyrin)

TM5 TM1; pESC-URA[GAL10/tHMGl]

TM7 TM3; pAG423[GALl/CYP716A021], pAG425[GALl/AtATRl]

TM8 TM3; pAG423[GALl/CYP716A021], pAG305[GALl/AtATRl]

TM9 TM3; pAG423[GALl/CYP716A021], pAG415[GALl/AtATRl]

TM10 TM3; pAG423[GALl/CYP716A12], pAG425[GALl/AtATRl]

TM17 TM3; pAG423[GALl/CYP716A12], pAG415[GALl/AtATRl]

TM21 TM3; pAG423[GALl/ML593], pAG415[GAZJ 4tA77?-!]

TM26 TM3; pAG423, pAG425[GALl/AtATRl]

TM30 TM3; pAG423[GALl/CYP716A021-T2A-CYP716A12], pAG415[G \i.-? i \r?-?]

TM27 TM3; pAG423, pAG415[G \i.-? i \r?-?]

TM31 TM3; pAG423[GALl/ML593-T2A-CYP716A12], pAG415[G \i.-? i \r?-?]

TM18 TM3; pAG423[GALl/CYP88D6], pAG415[G \i.-? i \r?-?] Name Construct

TM32 TM3; pAG423[GALl/ML593-T2A-CYP88D6], pAG415[GALl/AtATRl]

TM33 TM1; pESC-U RA[GAL10/tHMGl; GALl/CaDDS]

TM37 TM33; pAG423[GALl/ML593], pAG415[GALl/AtATRl]

TM38 TM33; pAG423, pAG415[GALl/AtATRl]

Table 5. Sequences of primers.

The sequences in lower case represent the restriction recognition site used for restriction enzyme mediated cloning. The underlined sequence corresponds to T2A partial sequences.

Table 6. List of sequences

SEQ ID NO

Nucleotide sequence

CYP716A021 1 ATGGAACTTTCTATCACTCTGATGCTTATTTTCTCAACAACCATCTTCT

TTATATTTCGTAATGTGTACAACCATCTCATCTCTAAACACAAAAACT

ATCCCCCTGGAAGTATGGGCTTGCCTTACATTGGCGAAACACTTAGTT

TCGCGAGATACATCACCAAAGGAGTCCCTGAAAAATTCGTAATAGAA

AGACAAAAGAAATATTCAACAACAATATTTAAGACCTCCTTGTTCGG

AG A A AAC ATG GTG GTGTTG G G C AGTG C AG AG G G C A AC AA ATTTATT

TTTGGAAGCGAGGAGAAGTATTTACGAGTGTGGTTTCCAAGTTCTGT

GGACAAAGTGTTCAAAAAATCTCATAAGAGAACGTCGCAGGAAGAA

G CT ATT AG GTTG CG C AAA AAC ATG GTG CC ATTTCTC AA AG C AG ATTT

GTTGAGAAGTTATGTACCAATAATGGACACATTTATGAAACAACATG

TGAACTCGCATTGGAATTGCGAGACCTTGAAGGCTTGTCCTGTGATC

AAGGA I 1 1 I ACG I 1 I AC I 1 1 AG I I 1 AAA I 1 1 1 1 1 1 1 AGTGTAGACA

ATCCTTTGGAGCTAGAGAAGTTAATCAAGCTATTTGTGAATATAGTG

AATGGCCTCCTTACGGTCCCTATTGATCTCCCGGGGACAAAATTTAGA SEQ ID NO

Nucleotide sequence

GGAGTTATAAAGAGTGTCAAGACTATTCGCCATGCGCTTAAAGTGTT

GATCAGGCAACGAAAGGTGGATATTAGAGAGAAAAGAGCCACACCT

ACGCAAGATATATTGTCGATAATGCTGGCACAGGCTGAGGACGAGA

ACTATGAAATGAATGATGAAGATGTGGCCAATGACTTTCTTGCAGTT

TTGCTTGCTAGTTATGATTCTGCCAATACTACACTCACCATGATTATG

AAATATCTTGCTGAATATCCCGAAATGTATGATCGAG 1 1 1 1 CAGAGA

ACAAATGGAGGTGGCAAAGACGAAAGGAAAAGATGAATTACTCAAC

TTGGACGACTTGCAAAAGATGAATTATACTTGGAATGTAGCTTGTGA

AGTACTGAGAATTGCAACACCAACGTTCGGAGCATTCAGAGAGGTTA

TTG C AG ATTGTAC ATACG AAG G GTAC ACC ATACC AA AAG G CTG G AA

GCTATATTATGCCCCGCG 1 1 1 1 ACCCATGGAAGTGCAAAATACTTTCA

AGATCCAGAGAAATTTGATCCATCGCGATTTGAAGGTGATGGTGCGC

CTCCTTATACATTCGTTCCATTCGGAGGAGGGCTCCGGATGTGCCCT

GGATACAAGTATGCAAAGATTATAGTACTAGTGTTCATGCACAATAT

AGTTACAAAGTTCAAATGGGAGAAAGTTAACCCTAATGAGAAAATG

ACAGTAGGAATCGTATCAGCGCCAAGTCAAGGACTTCCACTGCGTCT

CCATCCCCACAAATCTCCATCTTAA

2 ATGTG G GTAGTG G G ATTA ATTG GTGTG G CTGTG GTA AC AATATTG AT

AACTCAGTATGTATACAAATGGAGAAATCCAAAGACTGTGGGTGTTC

TGCCACCTGGTTCAATGGGTCTGCCTTTGATCGGGGAGACTCTTCAA

CTTCTCAGCCGTAATCCATCCTTGGATCTTCATCCTTTCATCAAGAGCA

GAATCCAAAGATATGGGCAGATATTCGCGACCAATATCGTAGGTCGA

CCCATAATAGTAACCGCTGATCCGCAGCTCAATAATTACC I 1 1 I CCAA

CAAGAAGGAAGAGCAGTAGAACTGTGGTACTTGGACAGCTTTCAAA

AGCTATTTAACTTAGAAGGTGCAAACAGGCCGAACGCAGTTGGTCAC

ATTCACAAGTACGTTAGAAGTGTATACTTGAGTCTCTTTGGCGTCGA

GAGCCTTAAAACAAAGTTGCTTGCCGATATTGAGAAAACAGTCCGCA

A AA ATCTT ATTG GTG G G AC AACC AA AG G C ACCTTTG ATG C AAA AC AT

G CTTCTG CC AATATG GTTG CTG 1 1 1 1 1 G CTG C AA AATACTTGTTCG G A

CATGATTACGAGAAATCGAAAGAAGATGTAGGCAGCATAATCGACA

ACTTCGTACAAGGTCTTCTCGCATTCCCATTGAATGTTCCCGGTACAA

AGTTCCACAAATGTATGAAGGACAAGAAAAGGCTGGAATCAATGAT

CACTAACAAGCTAAAGGAGAGAATAGCTGATCCGAACAGCGGACAA

GGGGATTTCCTTGATCAAGCAGTGAAAGACTTGAATAGCGAATTCTT

CATAACAGAGAC 1 1 1 1 ATCGTTTCGGTGACGATGGGAGCTTTATTTGC

GACGGTTGAATCGGTTTCGACAGCAATTGGACTAGCTTTCAAG 1 1 1 1 1

TGCAGAGCACCCCTGGG 1 1 1 1 GGATGACCTCAAGGCTGAGCATGAG

GCTGTCCTTAGCAAAAGAGAGGATAGAAATTCACCTCTCACGTGGGA

CGAATATAGATCGATGACACACACGATGCACTTTATCAATGAAGTCG

TCCGTTTGGGAAATG 1 1 1 1 I I GAA I 1 1 1 GAGGAAAGCACTGAAA

GATATTCCATATAATGGTTATACAATTCCGTCCGGTTGGACCATTATG

ATTGTGACCTCTACCCTTGCGATGAACCCTGAGATATTCAAGGATCCT

CTTGCATTCAATCCGAAACGTTGGCGGGATATTGATCCCGAAACTCA

A ACTAA A AACTTTATG CCTTTCG GTG GTG G G ACG AG AC A ATG CG C AG

GTG C AG AG CTAG CC AAG G C ATTCTTTG CTACCTTCCTCC ATG 1 1 1 I AA

TCAGCGAATATAGCTGGAAGAAAGTGAAGGGAGGAAGCGTTGCTCG

GACACCTATGTTAAG 1 1 1 1 GAAGATGGCATATTTATTGAGGTCACCAA

GAAAAACAAGTGA

Amino acid sequence SEQIDNO

Nucleotide sequence

CYP716A021 3 MELSITLMLIFSTTIFFIFRNVYNHLISKHKNYPPGSMGLPYIGETLSFARYI

TKGVPEKFVIERQKKYSTTIFKTSLFGENMVVLGSAEGNKFIFGSEEKYLR

VWFPSSVDKVFKKSHKRTSQEEAIRLRKNMVPFLKADLLRSYVPIMDTF

MKQHVNSHWNCETLKACPVIKDFTFTLACKLFFSVDNPLELEKLIKLFVNI

VNGLLTVPIDLPGTKFRGVIKSVKTIRHALKVLIRQRKVDIREKRATPTQDI

LSIMLAQAEDENYEMNDEDVANDFLAVLLASYDSANTTLTMIMKYLAE

YPEMYDRVFREQMEVAKTKGKDELLNLDDLQKMNYTWNVACEVLRIA

TPTFGAFREVIADCTYEGYTIPKGWKLYYAPRFTHGSAKYFQDPEKFDPS

RFEGDGAPPYTFVPFGGGLRMCPGYKYAKIIVLVFMHNIVTKFKWEKVN

PNEKMTVGIVSAPSQGLPLRLHPHKSPS

ML593 4 MWVVGLIGVAVVTILITQYVYKWRNPKTVGVLPPGSMGLPLIGETLQLL

SRNPSLDLHPFIKSRIQRYGQIFATNIVGRPIIVTADPQLNNYLFQQEGRA

VELWYLDSFQKLFNLEGANRPNAVGHIHKYVRSVYLSLFGVESLKTKLLA

DIEKTVRKNLIGGTTKGTFDAKHASANMVAVFAAKYLFGHDYEKSKEDV

GSIIDNFVQGLLAFPLNVPGTKFHKCMKDKKRLESMITNKLKERIADPNS

GQGDFLDQAVKDLNSEFFITETFIVSVTMGALFATVESVSTAIGLAFKFFA

EH PWVLDDLKAEHEAVLSKREDRNSPLTWDEYRSMTHTMHFI NEVVRL

GNVFPGILRKALKDIPYNGYTIPSGWTIMIVTSTLAMNPEIFKDPLAFNPK

RWRDIDPETQTKNFMPFGGGTRQCAGAELAKAFFATFLHVLISEYSWK

KVKGGSVARTPMLSFEDGIFIEVTKKNK

REFERENCES

Arthington-Skaggs, B.A., Jradi, H., Desai, T., and Morrison, C.J. (1999). Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. Journal of Clinical Microbiology 37, 3332-3337.

Asadollahi, M.A., Maury, J., M0ller, K., Nielsen, K.F., Schalk, M., Clark, A., and Nielsen, J. (2008).

Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis, biotechnology and bioengineering 99, 666-677.

Ashour, M.L., and Wink, M. (2011). Genus Bupleurum: a review of its phytochemistry, pharmacology and modes of action. Journal of Pharmacy and Pharmacology 63, 305-321.

Augustin JM, Kuzina V, Andersen SB, Bak S. Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry. 2011 Apr;72(6):435-57. Epub 2011 Feb 16.

Burnouf-Radosevich, M., Delfel, N.E., and England, R. (1985). Gas chromatography-mass sepctrometry of oleanane- and ursane type triterpones - application to Chenopodium Quinoa triterpenes. Phytochemistry 24, 2063-2066.

Carelli, M., Biazzi, E., Panara, F., Tava, A., Scaramelli, L, Porceddu, A., Graham, N., Odoardi, M., Piano, E., Arcioni, S., May, S., Scotti, C, and Calderini, O. (2011). Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins. The Plant Cell 23, 3070-3081.

de Felipe, P., Luke, G.A., Hughes, L.E., Gani, D., Halpin, C, and Ryan, M.D. (2006). E unum pluribus: multiple proteins from a self-processing polyprotein. Trends in Biotechnology 24, 68-75.

Faizal, A., Lambert, E., Foubert, K., Apers, S., and Geelen, D. (2011). In vitro propagation of four

saponin producing Maesa species. Plant Cell Tissue and Organ Culture 106, 215-223.

Foubert, K., Cuyckens, F., Vleeschouwer, K., Theunis, M., Vlietinck, A., Pieters, L., and Apers, S.

(2010). Rapid quantification of 14 saponins of Maesa lanceolatab UPLC-MS/MS. Talanta 81,

1258-1263.

Fujita, S., Ohnishi, T., Watanabe, B., Yokota, T., Takatsuto, S., Fujioka, S., Yoshida, S., Sakata, K., and

Mizutani, M. (2006). Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols. The Plant Journal 45, 765-774.

Guo, S.J., Kenne, L, Lundgren, L.N., Ronnberg, B., and Sundquist, B.G. (1998). Triterpenoid saponins from Quillaja saponaria. Phytochemistry 48, 175-180.

Fukushima, E.O., Seki, H., Ohyama, K., Ono, E., Umemoto, N., Mizutani, M., Saito, K., and Muranaka,

T. (2011). CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis. Plant and Cell Physiology 52, 2050-2061.

Fukushima, E.O., Seki, H., Sawai, S., Suzuki, M., Ohyama, K., Saito, K., and Muranaka, T. (2013).

Combinatorial biosynthesis of legume natural and rare triterpenoids in engineered yeast. Plant and Cell Physiology. (doi:10.1093/pcp/pct015).

Han, J.Y., Kim, H.J., Kwon, Y.S., and Choi, Y.E. (2011). The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-ll during ginsenoside biosynthesis in Panax ginseng. Plant and Cell Physiology 52, 2062-2073.

Han, J.Y., Hwang, H.S., Choi, S.W., Kim, H.J., and Choi, Y.E. (2012). Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng. Plant and Cell Physiology.

Hayashi, H., Huang, P., Kirakosyan, A., Inoue, K., Hiraoka, N., Ikeshiro, Y., Kushiro, T., Shibuya, M., and Ebizuka, Y. (2001). Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice. Biological and Pharmaceutical

Bulletin 24, 912-916.

Herrera, J.B., Bartel, B., Wilson, W.K., and Matsuda, S.P. (1998). Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene. Phytochemistry 49, 1905-1911. Huang, L, Li, J., Ye, H., Li, C, Wang, H., Liu, B., and Zhang, Y. (2012). Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus. Planta.

Huhman DV, Sumner LW. Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer. Phytochemistry. 2002 Feb;59(3):347-60.

Huhman DV, Berhow MA, Sumner LW. Quantification of saponins in aerial and subterranean tissues of

Medicago truncatula. J Agric Food Chem. 2005 Mar 23;53(6):1914-20.

Iturbe-Ormaetxe I, Haralampidis K, Papadopoulou K, Osbourn AE. Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus. Plant Mol Biol. 2003 Mar;51(5):731-43.

Kim, O.T., Kim, M.Y., Huh, S.M., Bai, D.G., Ahn, J.C., and Hwang, B. (2005). Cloning of a cDNA probably encoding oxidosqualene cyclase associated with asiaticoside biosynthesis from Centella asiatica (L.) Urban. Plant Cell Reports 24,304-311.

Kim, O.T., Lee, J.W., Bang, K.H., Kim, Y.C., Hyun, D.Y., Cha, S.W., Choi, Y.E., Jin, M.L., and Hwang, B.

(2009). Characterization of a dammarenediol synthase in Centella asiatica (L.) Urban. Plant

Physiology and Biochemistry 47, 998-1002.

Kirby, J., Romanini, D.W., Paradise, E.M., and Keasling, J.D. (2008). Engineering triterpene production in Saccharomyces cerevisiae -β-amyrin synthase from Artemisia annua. The FEBS Journal 275,

1852-1859.

Kunii, M., Kitahama, Y., Fukushima, E.O., Seki, H., Muranaka, T., Yoshida, Y., and Aoyama, Y. (2012). β-Amyrin oxidation by oat CYP51H10 expressed heterologously in yeast cells: the first example of CYP51-dependent metabolism other than the 14-demethylation of sterol precursors. Biological and Pharmaceutical Bulletin 35, 801-804.

Kushiro T, Shibuya M, Ebizuka Y. Beta-amyrin synthase-cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur J Biochem.

1998 Aug 15;256(l):238-44.

Li, L., Cheng, H., Gai, J., and Yu, D. (2007). Genome-wide identification and characterization of putative cytochrome P450 genes in the model legume Medicago truncatula. Planta 226, 109-123.

Manguro, L.O.A., Midiwo, J.O., Tietze, L.F., and Hao, P. (2011). Triterpene saponins of Maesa

lanceolata leaves. Arkivoc, 172-198.

Morita M, Shibuya M, Kushiro T, Masuda K, Ebizuka Y. Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum) new alpha-amyrin-producing enzyme is a multifunctional triterpene synthase. Eur J Biochem. 2000 Jun;267(12):3453-60.

Polakowski, T., Stahl, U., and Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology

49, 66-71.

Pollier, J., Morreel, K., Geelen, D., and Goossens, A. (2011). Metabolite profiling of triterpene saponins in Medicago truncatula hairy roots by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry. Journal of Natural Products 74, 1462-1476.

Pollier, J., Goossens, A. (2012). Oleanolic acid. Phytochemistry 77, 10-15.

Pollier, J., Gonzalez-Guzman, M., Ardiles-Diaz, W., Geelen, D., and Goossens, A. (2011b). An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences. PLoS ONE 6, e24978.

Reed, J.R., and Backes, W.L. (2012). Formation of P450 · P450 complexes and their effect on P450 function. Pharmacology & Therapeuctics 133, 299-310.

Rickling, B., and Glombitza, K.W. (1993). Saponins in the leaves of birch? Hemolytic dammarane

triterpenoid esters of Betula pendula. Planta medica 59, 76-79.

Rischer, H., Oresic, M., Seppanen-Laakso, T., Katajamaa, M., Lammertyn, F., Ardiles-Diaz, W., Van Montagu, M.C., Inze, D., Oksman-Caldentey, K.M., and Goossens, A. (2006). Gene-to- metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. Proceedings of the National Academy of Sciences of the United States of America 103, 5614- 5619.

Seki, H., Ohyama, K., Sawai, S., Mizutani, M., Ohnishi, T., Sudo, H., Akashi, T., Aoki, T., Saito, K., and Muranaka, T. (2008). Licorice beta-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proceedings of the National Academy of Sciences of the United States of America 105, 14204-14209.

Seki, H., Sawai, S., Ohyama, K., Mizutani, M., Ohnishi, T., Sudo, H., Fukushima, E.O., Akashi, T., Aoki, T., Saito, K., and Muranaka, T. (2011). Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin. Plant Cell 23, 4112- 4123.

Sabater-Jara Sabater-Jara AB, Almagro L, Belchi-Navarro S, Ferrer MA, Barcelo AR, Pedreno MA (2010). Induction of sesquiterpenes, phytoesterols and extracellular pathogenesis-related proteins in elicited cell cultures of Capsicum annuum. J Plant Physiol. 167:1273-81.

Sindambiwe, J.B., Calomme, M., Geerts, S., Pieters, L, Vlietinck, A.J., and VandenBerghe, D.A. (1998).

Evaluation of biological activities of triterpenoid saponins from Maesa lanceolate/. Journal of

Natural Products 61, 585-590.

Sparg SG, Light ME, van Staden J. Biological activities and distribution of plant saponins. J Ethnopharmacol. 2004 Oct;94(2-3):219-43.

Spencer, G.F. (1981). Dammarenediol II esters from Cacalia atriplicifolia L. seed oil. Journal of Natural Products 44, 166-168.Stiti, N., Triki, S., and Hartmann, M.A. (2007). Formation of triterpenoids throughout Olea europaea fruit ontogeny. Lipids 42, 55-67.

Sun HX, Xie Y, Ye YP. Advances in saponin-based adjuvants. Vaccine. 2009 Mar 13;27(12):1787-96.

Epub 2009 Feb 7.

Suzuki H, Achnine L, Xu R, Matsuda SP, Dixon RA. A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula. Plant J. 2002 Dec;32(6):1033-48.

Tava, A., Scotti, C, and Avato, P. (2011). Biosynthesis of saponins in the genus Medicago.

Phytochemistry Reviews 10, 459-469.

Toivounen L. (1993) Biotechnol. Prog. 9, 12.

Turpen TH. Tobacco mosaic virus and the virescence of biotechnology. Philos Trans R Soc Lond B Biol Sci. 1999 Mar 29;354(1383):665-73.

Vanhala L. et al. (1995) Plant Cell Rep. 14, 236.

Verpoorte, R. et al. (1999). Biotechnol. Lett. 21,467.

Vincken JP, Heng L, de Groot A, Gruppen H. Saponins, classification and occurrence in the plant kingdom. Phytochemistry. 2007 Feb;68(3):275-97.

Vuylsteke, M., Peleman, J.D., and van Eijk, M.J. (2007). AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis. Nat. Protoc. 2, 1399-1413.

Zou, K., Zhu, S., Tohda, C, Cai, S., and Komatsu, K. (2002). Dammarane-type triterpene saponins from

Panax japonicus. Journal of Natural Products 65, 346-351.