WO2024047057A1 - Means and methods to produce triterpene saponins in eukaryotic cells - Google Patents

Abstract

The present invention provides novel chimeric genes for the production of quillaic acid and derivatives thereof in eukaryotic cells such as yeasts and plants.

Description

MEANS AND METHODS TO PRODUCE TRITERPENE SAPONINS IN EUKARYOTIC CELLS

Field of the invention

The present invention relates to the field of plant secondary metabolites, more specifically to the field of saponins, even more specifically to the field of quillaic acid and derivatives thereof. The present invention provides chimeric genes and expression vectors for producing quillaic acid in native and heterologous host cells, such as plants and yeasts.

Introduction to the invention

Saponins, glycosides widely distributed in the plant kingdom, include a diverse group of compounds characterized by their structure containing a steroidal or triterpenoid aglycone and one or more sugar chains. Their structural diversity is reflected in their physicochemical and biological properties, which are exploited in a number of traditional (as soaps, fish poison, and molluscicides) and industrial applications. Recent research has established saponins as the active components in many herbal medicines and highlighted their contributions to the health benefits of foods such as soybeans and garlic. Saponins are glycosides containing one or more sugar chains on a triterpene or steroid aglycone backbone also called a sapogenin. They are categorized according to the number of sugar chains in their structure as mono, bi-, or tridesmosidic. Monodesmosidic saponins have a single sugar chain, normally attached at C-3. Bidesmosidic saponins have two sugar chains, often with one attached through an ether linkage at C-3 and one attached through an ester linkage at C-28 (triterpene saponins) or an ether linkage at C-26 (furastanol saponins). The most common monosaccharides include: D-glucose (Glc), D-galactose (Gal), D-glucuronic acid (GlcA), D-galacturonic acid (GalA), L-rhamnose (Rha), L-arabinose (Ara), D-xylose (Xyl), and D-fucose (Fuc). The nature of the aglycone and the functional groups on the aglycone backbone and number and nature of the sugars can vary greatly resulting in a very diverse group of compounds.

Quillaic acid is the major aglycone of the widely studied saponins of the Chilean indigenous tree Quillaja saponaria. Several glucoside modified variants of quillaic acid are known. One is the saponin QS-21 which is known as a potent adjuvant for CTL induction, and induces Thl cytokines (see Kensil CR et al (1995) in Powel, MF, Newman, MJ (Eds), Vaccine Design, p. 525-541). Another glucoside modified variant is sapofectosid (also known as SO1861) which is known as a transfection reagent (see Sama S et al (2017) Int. J. of Pharmaceutics 534, 195-205) and a potent endosomal escape molecule. Quillaja saponins and the extensive functionalization of this triterpenoid provide unique opportunities for structural modification and pose a challenge from the standpoint of selectivity in regard to one or the other secondary alcohol group, the aldehyde, and the carboxylic acid functions. The availability of quillaic acid and its modified variants is limited and chemical synthesis is cumbersome and expensive. It would be an advantage to be able to produce quillaic acid and modified versions thereof in eukaryotic organisms such as for example yeasts and plants. WO2019/122259 describes the isolation of nucleic acid sequences encoding biosynthetic enzymes from Quillaja saponaria which when expressed in heterologous hosts can produce precursors of quillaic acid. The instant invention has isolated alternative enzymes from Saponaria officinalis which can synthesize quillaic acid in heterologous eukaryotic hosts such as yeasts. Interestingly two of the isolated nucleic acids, from the instant invention, each of them when overexpressed as a chimeric gene in hairy roots of Saponaria lead to the production of sapofectosid (SO1861).

Legends to the figures

Figure 1: Expression trends for selected transcripts. The left and right panels depict data as scaled expression values, and transcript abundances as Counts Per Million (CPM)

Figure 2: Agarose gel electrophoresis showing DNA bands corresponding to amplified target genes from cDNA. Target genes are reported within the grey boxed below the corresponding gel bands. J = Jasmonate, 6 = six hours after treatment, L = leaves, R = roots, P = plantlets.

Figure 3: GC-MS analysis revealed that all strains, except the negative control, accumulated a peak at 15.48 corresponding to p-amyrin - results are shown for strain 1, 2, and 3.

Figure 4: recombinant yeast strains 4, 5 and 6 expressed a single P450 (either SoP450_1944, SoP450_2010 or SoP450_1049 respectively) together with tHMGRl, GgBAS and MtMTRl. Recombinant yeast strain 4 (SoP450_1944) was found to accumulate erythrodiol (17.27 min), the C-28 alcohol of p- amyrin in the pellet.

Figure 5: recombinant yeast strains 4, 5 and 6 expressed a single P450 (either SoP450_1944, SoP450_2010 or SoP450_1049 respectively) together with tHMGRl, GgBAS and MtMTRl. Recombinant yeast strain 4 (SoP450_1944) was found to accumulate oleanolic acid (18.37 min) and all its intermediates derived from C-28 p-amyrin oxidation such as erythrodiol (17.25 min) and oleanolic aldehyde (18.83 min).

Figure 6: Recombinant yeast strain 6 (expressing SoP450_1049) didn't produce any additional peak in the pellet (see Fig. 4), whereas in the medium erythrodiol (17.25 min), oleanolic acid (18.37 min) and echinocystic acid (19.00 min) were detected. The latter compound corresponds to 16-hydroxy-oleanolic acid and therefore leads to the identification of SoP450_1049 as an enzyme with a dual function as a C- 28/C-16 oxidase. Figure 7: When the two enzymes SoP450_1944 and SoP450_2010 were combined in recombinant yeast strain 7 , no additional products were detected in the cell pellet, though the levels of erythrodiol (17.27 min) and 23-hydroxy-p-amyrin (16.66 min) were notably reduced.

Figure 8: Analysis of liquid medium of recombinant yeast strain 7 revealed that, besides the products already detected in yeast strains 4 (SoP450_1944) and 5 (SoP450_2010) expressing each of these two P450s individually, gypsogenin (22.53 min) and gypsogenic acid (23.44 min) were present as additional products. Both of these compounds result from a C-23 oxidation of oleanolic acid: gypsogenin is characterized by aldehyde function at position 23 while the same carbon is further oxidized to carboxy group in gypsogenic acid.

Figure 9: Combination of SoP450_1944 and SoP450_1049 in strain 8 did not lead to accumulation of any additional products in the pellet as compared to the yeast strains expressing the same enzymes individually. This is because SoP450_1049 already appeared to have both C-28 and C-16 oxidase activity.

Figure 10: Combination of SoP450_1944 and SoP450_1049 in strain 8 did not lead to accumulation of any additional products in liquid medium as compared to the yeast strains expressing the same enzymes individually. This is because SoP450_1049 already appeared to have both C-28 and C-16 oxidase activity.

Figure 11: No additional products were detected in the pellet of recombinant yeast strain 9 co-expressing SoP450_2010 and SoP450_1049, as compared to strains 5 (SoP450_2010) and 6 (SoP450_1049) expressing each enzyme individually.

Figure 12: No additional products were detected in the medium of yeast strain 9 co-expressing SoP450_2010 and SoP450_1049, as compared to strains 5 (SoP450_2010) and 6 (SoP450_1049) expressing each enzyme individually.

Figure 13: When the three P450 enzymes SoP450_1944, SoP450_1049 and SoP450_2010 were coexpressed together, in recombinant yeast strain 10, no new peaks appeared in the cell pellet although P-amyrin (15.38 min) was reduced, thus suggesting further conversion. This was confirmed by analysis of the yeast medium for the same strain, which showed a new peak (22.67 min) detected only in strain 10.

Figure 14: No additional peaks were identified in recombinant strain 16 by concomitant expression of SoP450_6085 and SoP450_1049, as compared to strain 6 expressing only SoP450_6085.

Figure 15: The most unexpected result was obtained by metabolite profiling of recombinant yeast strain 19 where the dual specificity C-28/C-16 oxidase (SoP450_1049) was co-expressed with both C-23 oxidases (SoP450_2010 and SoP450_6085). This recombinant yeast strain, beside all the products derived from C-28 and C-16 oxidation of p-amyrin (such as erythrodiol, oleanolic and echinocystic acid), was also found to synthesize 16-hydroxy-gypsogenic acid (similarly to recombinant yeast strain 10) but differently it also accumulated quillaic acid and traces of gypsogenic acid.

Figure 16: content of sapofectosid in S. officinalis hairy root lines generated in this invention

Figure 17: Schematic representation of the triterpene aglycone biosynthetic pathway in S. officinalis

Figure 18: content of selected saponins in G. elegans hairy root lines generated in this invention. Normalized intensities have been obtained by dividing the peak are of the compound of interest by the peak are of internal standard.

Detailed description of the invention

To facilitate the understanding of this invention a number of terms are defined below. Terms defined herein (unless otherwise specified) have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. As used in this specification and its appended claims, terms such as "a", "an" and "the" are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration, unless the context dictates otherwise. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

In the present invention we have identified biosynthetic enzymes in Saponaria officinalis, which enzymes when expressed as chimeric genes in an eukaryotic host, lead to the production of quillaic acid. We have surprisingly shown that for the first time quillaic acid can be produced in the yeast Saccharomyces cerevisiae by expression of the identified chimeric genes. Even more surprisingly the isolated biosynthetic enzymes comprise an unusual combination of a dual specificity C-16/C-28 oxidase and two different C-23 oxidases. Even more surprisingly the overexpression of only a chimeric gene encoding the isolated beta-amyrin synthase or only a chimeric gene encoding the C-28/C-16 oxidase in Saponaria hairy roots results in an increase of SO1861 (or sapofectosid) levels in said hairy roots.

Accordingly, the present invention provides in a first embodiment a eukaryotic cell comprising at least one of the following chimeric genes: a. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 2 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 2 and transcription termination and polyadenylation signals, b. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 6 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 6 and transcription termination and polyadenylation signals, c. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 8 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 8 and transcription termination and polyadenylation signals, d. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 10 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 10 and transcription termination and polyadenylation signals.

In yet another embodiment the eukaryotic cell further comprises a chimeric gene encoding an N- terminal truncated feedback-insensitive 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Non-limiting examples of N-terminal truncated feedback-insensitive 3-hydroxy-3-methylglutaryl coenzyme A reductases are depicted in the nucleotide sequences SEQ ID NO: 13 and 19.

In yet another embodiment the eukaryotic cell further comprises a chimeric gene encoding a NADPH- cytochrome P450 reductase. A non-limiting example of a NADPH-cytochrome P450 oxidase is depicted in the nucleotide sequence SEQ ID NO: 15.

It is understood that eukaryotic cells can be higher or low eukaryotic cells. Higher eukaryotic cells comprise plant cells and animal cells. Lower eukaryotic cells comprise yeast and fungal cells. Yeast can for example be from the genus Saccharomyces, Pichia, Yarrowia, Hansenula or Kluyveromyces. Fungal cells can for example be from the genus Aspergillus or Trichoderma.

SEQ ID NO: 2 is a beta-amyrin synthase isolated from Saponaria officinalis.

SEQ ID NO: 6 is a combined C-28/C-16 oxidase of beta-amyrin isolated from Saponaria officinalis.

SEQ ID NO: 8 and 10 are two different C-23 oxidases of beta-amyrin isolated from Saponaria officinalis.

In another embodiment the invention provides a chimeric gene construct comprising the following operably linked DNA elements: a) a promoter region which is active in plant or yeast cells, b) a DNA region encoding SEQ ID NO: 6 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 6 and c) a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant or yeast.

In another embodiment the invention provides a chimeric gene construct comprising the following operably linked DNA elements: a) a promoter region which is active in eukaryotic cells such as plant or yeast cells, b) a DNA region encoding SEQ ID NO: 8 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 8 and c) a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant or yeast.

In another embodiment the invention provides a chimeric gene construct comprising the following operably linked DNA elements: a) a promoter region which is active in yeast or plant cells, b) a DNA region encoding SEQ ID NO: 10 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 10 and c) a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of yeast or plants.

In another embodiment the invention provides a yeast cell which yeast cell comprises the following chimeric genes: a. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 2 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 2 and a terminator sequence, b. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 6 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 6 and a terminator sequence, c. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 8 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 8 and a terminator sequence, d. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 10 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 10 and a terminator sequence.

In yet another embodiment the yeast cell further comprises a chimeric gene encoding an amino-terminal truncated feedback-insensitive 3-hydroxy-3-methylglutaryl coenzyme A reductase.

In yet another embodiment the yeast cell further comprises a chimeric gene encoding an amino-terminal truncated feedback-insensitive 3-hydroxy-3-methylglutaryl coenzyme A reductase and a chimeric gene encoding a NADPH-cytochrome P450 reductase.

In yet another embodiment the invention provides a plant cell comprising a chimeric gene comprising a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 2 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 2 and a terminator sequence, or said plant cell comprises a chimeric gene comprising a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 6 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 6 and a terminator sequence. In a particular embodiment said plant cell is from the genus Saponaria or Gypsophila.

By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA).

In a particular embodiment, the promoter is an inducible promoter.

The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.

Thus, a nucleic acid according to the invention may be placed under the control of an externally (inducible) gene promoter to place expression under the control of the user. An advantage of introduction of a heterologous gene into a plant or yeast cell, is the ability to place expression of the gene under the control of a promoter of choice, in order to be able to influence gene expression, and therefore quillaic or sapofectosid, according to preference.

Suitable promoters which operate in plants include the Cauliflower Mosaic Virus 35S (CaMV 35S). Other examples are disclosed at pg. 120 of Lindsey & Jones (1989)"Plant Biotechnology in Agriculture" Pub. OU Press, Milton Keynes, UK. The promoter may be selected to include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression. Inducible plant promoters include the ethanol induced promoter of Caddick et al (1998) Nature Biotechnology 16: 177-180. Suitable yeast promoters are for example the galactose inducible promoter from Saccharomyces cerevisiae or the methanol-inducible promoters from Pichia pastoris.

In yet another embodiment the invention provides a method to produce quillaic acid in yeast comprising introducing the following chimeric genes: a. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 2 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 2 and a polyadenylation and termination sequence, b. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 6 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 6 and a polyadenylation and termination sequence, c. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 8 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 8 and a polyadenylation and termination sequence, d. a yeast-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 10 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 10 and a polyadenylation and termination sequence, cultivating said transformed yeast and isolating quillaic acid from said cultivated yeast.

In yet another embodiment the method to produce quillaic acid in yeast further comprises the introduction of a chimeric gene encoding an amino-terminal truncated feedback-insensitive 3-hydroxy- 3-methylglutaryl coenzyme A reductase.

In yet another embodiment the method to produce quillaic acid in yeast further comprises the introduction of a chimeric gene encoding a NADPH-cytochrome P450 reductase.

In yet another embodiment the invention provides a method to produce sapofectosid in Saponaria or Gypsophila species comprising introducing the chimeric gene: a. a plant-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 2 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 2 and transcription termination and polyadenylation signals, or the chimeric gene b. a plant-expressible promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 6 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 6 and transcription termination and polyadenylation signals, by transformation into hairy roots of a Saponaria or Gypsophila species, cultivating said transformed hairy roots and isolating sapofectosid from said cultivated transformed hairy roots.

The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest or a homologue thereof as defined herein above.

A "chimeric gene" or "chimeric construct" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence and a terminator sequence. The regulatory nucleic acid sequence of the chimeric gene is not normally operatively linked to the associated nucleic acid sequence as found in nature.

The term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant or yeast genes, or from T-DNA. For plant terminators, the terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene.

"Selectable marker", "selectable marker gene" or "reporter gene" includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid construct of the invention. These marker genes enable the identification of a successful transfer of the nucleic acid molecules via a series of different principles. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptll that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin, or genes conferring resistance to, for example, bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin), to herbicides (for example bar which provides resistance to Basta*; aroA or gox providing resistance against glyphosate, or the genes conferring resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source or xylose isomerase for the utilisation of xylose, or antinutritive markers such as the resistance to 2-deoxyglucose). Expression of visual marker genes results in the formation of colour (for example p-glucuronidase, GUS or p- galactosidase with its coloured substrates, for example X-Gal), luminescence (such as the luciferin/luciferase system) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). This list represents only a small number of possible markers. The skilled worker is familiar with such markers. Different markers are preferred, depending on the organism and the selection method.

It is known that upon stable or transient integration of nucleic acids into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).

Since the marker genes, particularly genes for resistance to antibiotics and herbicides, are no longer required or are undesired in the transgenic host cell once the nucleic acids have been introduced successfully, the process according to the invention for introducing the nucleic acids advantageously employs techniques which enable the removal or excision of these marker genes. One such a method is what is known as co-transformation. The co- transformation method employs two vectors simultaneously for the transformation, one vector bearing the nucleic acid according to the invention and a second bearing the marker gene(s). A large proportion of transformants receives or, in the case of plants, comprises (up to 40% or more of the transformants), both vectors. In case of transformation with Agrobacteria, the transformants usually receive only a part of the vector, i.e. the sequence flanked by the T-DNA, which usually represents the expression cassette. The marker genes can subsequently be removed from the transformed plant by performing crosses. In another method, marker genes integrated into a transposon are used for the transformation together with desired nucleic acid (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are transformed with a nucleic acid construct conferring expression of a transposase, transiently or stable. In some cases (approx. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost. In a further number of cases, the transposon jumps to a different location. In these cases the marker gene must be eliminated by performing crosses. In microbiology, techniques were developed which make possible, or facilitate, the detection of such events. A further advantageous method relies on what is known as recombination systems; whose advantage is that elimination by crossing can be dispensed with. The best-known system of this type is what is known as the Cre/lox system. Crel is a recombinase that removes the sequences located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed once transformation has taken place successfully, by expression of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration into the plant genome of the nucleic acid sequences according to the invention is possible. For the purposes of the invention, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention.

A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not present in, or originating from, the genome of said plant, or are present in the genome of said plant but not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.

For the purpose of this invention related or orthologous genes of the genes as described herein before can be isolated from the (publicly) available sequence databases. The "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xlOO) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J Mol Biol. 48: 443-453) The computer-assisted sequence alignment above, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wisconsin, USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3. Sequences are indicated as "essentially similar" when such sequence have a sequence identity of at least about 75%, particularly at least about 80 %, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical.

The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific genetic construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

The term "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. 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 leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), hairy roots and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363- 373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1 102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491 - 506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al (1984) Nucl. Acids Res. 12-8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agro bacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:1 -9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289], Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551 -558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316: 1 194-1 199], while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743], A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229], Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).

The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or Tl) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and nontransformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

The term "plant" as used herein encompasses whole monocotyledonous and dicotyledonous plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term "plant" also encompasses monocotyledonous and dicotyledonous plant cells, suspension cultures, callus tissue, hairy roots, embryos, meristematic regions, gametophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.

The term "expression cassette" refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including, in addition to plant cells, prokaryotic, yeast, fungal, insect or mammalian cells. The term includes linear and circular expression systems. The term includes all vectors. The cassettes can remain episomal or integrate into the host cell genome. The expression cassettes can have the ability to selfreplicate or not (i.e., drive only transient expression in a cell). The term includes recombinant expression cassettes that contain only the minimum elements needed for transcription of the recombinant nucleic acid.

The following non-limiting Examples describe methods and means according to the invention. Unless stated otherwise in the Examples, all techniques are carried out according to protocols standard in the art. The following examples are included to illustrate embodiments of the invention. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Examples and materials and methods

Example 1: Identification of novel enzymes involved in saponin biosynthesis in Saponaria officinalis

To identify the genes encoding the enzymes involved in the biosynthesis of triterpene saponins in Saponaria officinalis (soapwort), co-expression analysis was conducted on S. officinalis transcriptome datasets produced in-house and comprising different organs, sampled in response to hormonal treatments at multiple time points. More specifically, leaves, stems and roots from hydroponically grown S. officinalis plants were sampled in triplicate at six and twenty-four hours after mock or 50 pM Methyl Jasmonate treatment. S. officinalis seeds were sourced from the seed company Jelitto (https://www.ielitto.com/, Schwarmstedt, Germany).

RNA sequencing datasets were used to draft an S. officinalis reference transcriptome as well as to conduct gene expression analysis. To this end, RNA was extracted from the sampled S. officinalis tissues using the ReliaPrep RNA miniprep Systems (Promega™), following manufacturer's instructions for fibrous tissues. RNA was single-end sequenced via Illumina HiSeq 6000 with single-end read lengths of 100 bp for gene expression profiling. RNA was also used as template for cDNA synthesis using qScript cDNA SuperMix (QuantaBio™) and for the cloning of candidate genes. The S. officinalis draft transcriptome was produced using public datasets available through the 1KP project [1] and derived from HiSeq paired-end sequencing of flowers, fruits, stems and leaves that were therefore assembled by CLC genomics workbench using default parameters. Transcripts levels were estimated by mapping single-end reads on the S. officinalis transcriptome using Salmon [2] implemented on a Galaxy pipeline. Next, transcripts that were lowly expressed or with zero expression variance across samples were removed and the remaining genes were clustered using Self Organizing Map (SOM) implemented and visualized in R using the Kohonen-package [3] to assemble clusters of transcripts characterized by similar expression trends. The expression trends and transcript abundances for the genes considered in this study that will be discussed below are reported in Figure 1.

1.1 P-amyrin synthase

Gene functional annotation on the S. officinalis transcriptome allowed identification, by sequence homology with previously characterized enzymes from other plants, of a candidate p-amyrin synthase (BAS, hereafter referred to as SoBAS_1421, see SEQ ID NO: 1 for the coding sequence) that was therefore selected as the main bait for co-expression analysis. BAS is the key enzyme that commits linear triterpene precursors towards biosynthesis of oleanane-type pentacyclic triterpenes via cyclization of 2,3- oxidosqualene. The SoBAS_1421 full coding sequence and predicted protein are reported as SEQ ID NOs: 1 and 2.

1.2 P-amyrin oxidases

Four full length-length transcripts corresponding to cytochrome P450s (P450s) were identified among the genes co-expressed with SoBAS_1421. P450s proteins belong to a broad superfamily of enzymes catalyzing oxidation of various substrates. Phylogenetic analysis was performed to infer the function of the four candidate P450s from S. officinalis based on the comparison of protein sequences with P450s from other plant species reported to be involved in the oxidation of triterpenes.

The first P450 (hereafter referred to as SoP450_1944, see SEQ ID NO: 3 for the coding sequence) showed phylogenetic relations with plant P450s belonging to the CYP716 family that had been characterized as P-amyrin C-28 oxidases, suggesting that SoP450_1944 may catalyze the same reaction in S. officinalis cells. The full coding sequence and predicted protein sequences for SoP450_1944 are reported as SEQ ID NOs: 3 and 4.

The second P450 (hereafter referred to as SoP450_1049, see SEQ ID NO: 7 for the coding sequence) showed sequence similarities with CYP716A141 from Panax ginseng, the latter being the only example reported so far of an enzyme capable of oxidizing the C-28 of position of -amyrin and the C-16 p position of oleanolic acid. The full coding sequence and predicted sequences for SoP450_1049 are reported as SEQ ID NOs: 5 and 6.

The third P450 identified (hereafter referred to as SoP450_2010, see SEQ ID NO: 5 for the coding sequence) was found to be neighboring P450s involved in oxidation of position C23 of pentacyclic triterpenes. The full nucleotide and predicted protein sequences of SoP450_2010 are reported as SEQ ID NOs: 7 and 8.

Also the fourth P450 identified (hereafter referred to as SoP450_6085, see SEQ ID NO: 9 for the coding sequence) was found to be neighboring P450s involved in oxidation of position C23 of pentacyclic triterpenes. The full nucleotide and predicted protein sequences of SoP450_6085 are reported as SEQ ID NOs: 9 and 10.

1.3 Accessory enzymes

The UDP-glycosyltransferase (UGT) encoding gene SoUGT_2488 (SEQ ID NOs: 17 and 18) was identified in S. officinalis by sequence homology with previously reported UGTs from other plant species [4] as a gene encoding for an enzyme involved in transferring the first UDP-sugar (UDP-Glucuronic Acid) on to the C-3 position of quillaic acid. Therefore it may play a role in committing flux of aglycones towards the biosynthesis of highly-glycosylated oleanane-type saponins. SotHMGR (SEQ ID NOs: 19 and 20) was instead identified and cloned as truncated feedback-insensitive version of 3-hydroxy-3-methylglutaryl coenzyme A reductase (tHMGR), an enzyme known to catalyze the rate-limiting step in the biosynthesis of triterpene precursors [5], Lastly the gene SoSQE2 (SEQ ID Nos: 21 and 22) was identified as squalene epoxidase, an enzyme known to catalyze the stereospecific conversion of squalene to 2,3(S)- oxidosqualene, a key precursor in triterpenoid saponins. Therefore, co-expression of SoSQE2 together with other triterpene biosynthetic enzymes can boost production of precursors and ultimately triterpenoids yield as previously reported (Dong L. et al (2018) Metab Eng. 49:1-12).

Example 2: Cloning candidate genes from S. officinalis and generation of expression constructs for functional analysis

Each of the seven target genes described above was amplified by PCR using specific primer sets (see Table 1). Each of the primers included attB adapter sequences at the 5' end to allow directional cloning in appropriate Gateway® vectors (indicated in red in Table 1).

Table 1: Primers used in the present invention

Two PCR reactions were performed on each gene using either leaf or root cDNA as a template. PCR reactions were set up in a total volume of 25 pl using Q5 polymerase (New England Biolabs) according to the manufacturer's instructions. For amplification of SoBAS_1421 and SoP450_1049, PCR thermal cycling involved initial denaturation at 98°C (30 sec) followed by 3 cycles of denaturation (98°C, 10 sec) annealing (61°C, 20 sec), extension (72°C, 1 min 10 sec), followed by 30 cycles using the same conditions but increasing the annealing temperature to 70° C, with a final extension at 72° C (5 min). To amplify SoP450_2010 and SoP450_1944, the amplification protocol was slightly modified involving initial denaturation at 98°C (30 sec) followed by 10 cycles of denaturation (98°C, 10 sec), annealing (52°C, 20 sec), and extension (72°C, 1 min 30 sec), then followed by 25 cycles using the same conditions but increasing the annealing temperature to 60° C, with a final extension at 72° C (5 min). The same conditions were used to amplify SoUGT_2488 and SotHMGR. ForSoP450_6085 thermal cycling was set up to include 98°C (30 sec) followed by 35 cycles of denaturation (98°C, 10 sec), annealing (60°C, 20 sec), extension (72°C, 40 sec), and a final extension at 72° C (5 min).

All genes were successfully amplified from both mock-treated leaf and root cDNA except for SoP450_6085 and SotHMGR, which were cloned from mock-treated root or whole-plantlet cDNA and cDNA derived from JA-treated roots, respectively (Fig. 2). For each gene, the PCR product obtained from roots was purified and recombined into Gateway entry vector pDONR221 according to the manufacturer's protocols. The resulting plasmids were transformed into E.coli DH5a, extracted and sequenced to verify presence of correct inserts. Afterwards, each gene in entry clones was further recombined into the appropriate destination vectors via LR cloning.

Example 3: Functional analysis of candidate enzymes by expression in yeast

For heterologous expression in yeast, five different expression vectors were selected that (i) allow inducible transgene expression upon switching from glucose to galactose as the carbon source in the culture medium and (ii) carry different auxotrophic markers. Therefore, SoBAS_1421 was cloned into pESC (uracil selection). This vector was modified to be used in gateway recombination systems, as described previously [6], The same plasmid also contained a feedback-insensitive truncated version of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (tHMGRl) from Medicago truncatula. tH MGR genes have been extensively reported to increase triterpene production upon transient or stable expression in heterologous as well as homologous systems [5], SoP450_1944, SoP450_2010 and SoP450_1049 were cloned into the Gateway-compatible yeast vectors pAG424GAL (tryptophan selection), pAG425GAL (leucine selection) and pAG427GAL (methionine selection), respectively. SoP450_6085 was cloned in all three different vectors (pAG424GAL, pAG425GAL and pAG427GAL) to allow combination with each of the others P450s. Finally, in order to increase the catalytic efficiency of the P450s, a partner reductase enzyme known as Cytochrome P450 reductase (MtMTRl - MTR_3gl00160, SEQ. ID NOs: 15 and 16) from M. truncatula was cloned into pAG423GAL (histidine selection). All vectors contain galactose-inducible promoters driving the expression of inserted genes.

All these plasmids were transformed in S. cerevisiae to assess the function of each gene individually or in different combinations, as described in table 2. The yeast strain used in this study was derived from BY4272 (genotype: MATa; his3Al; leu2A0; ura3A0; lys2A0; trplAO; metl5A0; PAHl-Ob; Perg7::PMET3- ERG7), which contains five auxotrophic selection markers (-URA/-HIS/-LEU/-MET/-TRP) and therefore allows expression of genes from up to five plasmids.

The transformed yeast strains were selected on solid synthetic yeast media with appropriate supplements. Selected yeast strains were cultured in synthetic liquid medium with Galactose as the only carbon source and incubated for 7 days at 30°C. Methyl-p-cyclodextrin were added to the liquid medium at day two and day four during cultivation to reach a final concentration of 10 mM. Methyl-P- cyclodextrins are cyclic oligosaccharides that are able to sequester apolar triterpenes from yeast cells into the liquid medium thus avoiding possible toxicity and feedback inhibition by pathway intermediates, and thereby increase their production yield [7], Strains were pelleted by centrifugation and metabolites were extracted, separately for cell pellets and liquid culture medium, by liquid-liquid separation using n- hexane and ethyl acetate. Organic phases were collected, lyophilized by vacuum and analyzed by GC- MS.

SoBAS 1421 is a monofunctional P-amyrin synthase from S. officinalis

GC-MS analysis revealed that all strains, except the negative control, accumulated a peak at 15.48 corresponding to p-amyrin (Fig. 3 - results are shown for strain 1, 2, and 3). It is worth to mention that except strain 3 (carrying SoBAS_1421) and strain 11 (the negative control carrying empty vectors), all other strains carried GgBAS, a reported p-amyrin synthase from Glycyrrhiza glabra (SEQ ID NOs: 11 and 12). Besides p-amyrin, no side products were detected in strain 3 (expressing SoBAS_1421) thus confirming that SoBAS_1421 is a cyclase encoding for p-amyrin synthase, p-amyrin was predominantly present in the yeast pellet and at a lesser extent in the growth medium.

Discovery of C-28, C-28/C-16 and C-23 oxidases

Strain 4, 5 and 6 expressed a single P450 (either SoP450_1944, SoP450_2010 or SoP450_1049 respectively) together with tHMGRl, GgBAS and MtMTRl. Strain 4 (SoP450_1944) was found to accumulate erythrodiol (17.27 min), the C-28 alcohol of p-amyrin in the pellet (Fig. 4), whereas the medium (Fig. 5) contained oleanolic acid (18.37 min) and all its intermediates derived from C-28 p-amyrin oxidation such as erythrodiol (17.25 min) and oleanolic aldehyde (18.83 min). This confirmed that SoP450_1944 corresponds to a p-amyrin C-28 oxidase. In both pellet and medium (Fig. 4 and 5) of strain 5 (SoP450_2010) , only a peak corresponding to 23-hydroxy-p-amyrin (16.66 min) was detected, confirming this enzyme as a C-23 oxidase. Differently, strain 13 (expressing SoP450_6085) was found to produce only p-amyrin showing that this C-23 oxidase (SoP450_6085) is not able to function on such compound suggesting that perhaps it accepts only substrates with a higher degree of oxidation (Fig. 5). Strain 6 (expressing SoP450_1049) didn't produce any additional peak in the pellet (Fig. 4), whereas in the medium (Fig. 6) erythrodiol (17.25 min), oleanolic acid (18.37 min) and echinocystic acid (19.00 min) were detected. The latter compound corresponds to 16-hydroxy-oleanolic acid and therefore leads to the identification of SoP450_1049 as an enzyme with a dual function as a C-28/C-16 oxidase. Concomitant expression of C28-oxidase and C-23 oxidase

When the two enzymes SoP450_1944 and SoP450_2010 were combined in strain 7 , no additional products were detected in the cell pellet (Fig. 7), though the levels of erythrodiol (17.27 min) and 23- hydroxy-p-amyrin (16.66 min) were notably reduced. Analysis of liquid medium of strain 7 (Fig. 8) revealed that, besides the products already detected in yeast strains 4 (SoP450_1944) and 5 (SoP450_2010) expressing each of these two P450s individual ly, gypsogenin (22.53 min) and gypsogenic acid (23.44 min) were present as additional products. Both of these compounds result from a C-23 oxidation of oleanolic acid: gypsogenin is characterized by aldehyde function at position 23 while the same carbon is further oxidized to carboxy group in gypsogenic acid.

These results show that SoP450_2010 is able to accept oleanolic acid as a substrate and confirm its activity to be specifically oxidizing the carbon at position 23. Unexpectedly, strains 14 (Fig. 13) expressing the second candidate C-23 oxidase (SoP450_6085) together with the C-28 oxidase (SoP450_1944) did not produce any additional compound from those produced by strain 4 expressing SoP450_1944 solely.

Concomitant expression of C28-oxidase and C-28/C-16 oxidase

Combination of SoP450_1944 and SoP450_1049 in strain 8 did not lead to accumulation of any additional products, either in the pellet or liquid medium (Fig. 9 and 10), as compared to the yeast strains expressing the same enzymes individually. This is due to the fact that SoP450_1049 already appeared to have both C-28 and C-16 oxidase activity.

Concomitant expression of C23-oxidase and C-28/C-16 oxidase

No additional products were detected, either in the pellet (Fig. 11) or the medium (Fig. 12) of yeast strain 9 co-expressing SoP450_2010 and SoP450_1049, as compared to strains 5 (SoP450_2010) and 6 (SoP450_1049) expressing each enzyme individually. However, levels of hydroxy 23-hydroxy- p-amyrin (16.66 min), erythrodiol (17.25 min), oleanolic acid (18.37 min) and echinocystic acid (19.00 min) were found to be notably reduced. Similarly no additional peaks were identified in strain 16 (Fig. 14) by concomitant expression of SoP450_6085 and SoP450_1049, as compared to strain 6 expressing solely the latter enzyme.

Concomitant expression of three P450s enzymes

Next, all possible combinations involving three of the four P450s discussed so far were expressed in yeast thus generating strains 10, 17, 18 and 19 (see Table 1).

When the three P450 enzymes SoP450_1944, SoP450_1049 and SoP450_2010 were expressed together, in yeast strain 10, no new peaks appeared in the cell pellet (Fig. 12) although -amyrin (15.38 min) was reduced, thus suggesting further conversion. This was confirmed by analysis of the yeast medium (Fig. 13) for the same strain, which showed a new peak (22.67 min) detected only in strain 10. Based on the observed enzyme activities of all three P450s, this peak would correspond to oxidation of 3-OH, 16-OH, 23-COOH and 28-COOH on the p-amyrin backbone, leading to a compound also called 16-hydroxy- gypsogenic acid. This was further confirmed after purification of the compound and structure elucidation by Nuclear Resonance Mass Spectrometry (data not shown).

Yeast strain 18 differed from yeast strain 10 by substitution of SoP450_2010 with another C-23 oxidase, SoP450_6085. Interestingly, and unexpectedly, this combination did not yield any new compound as compared to combination 8 (expressing SoP450_1944 and SoP450_1049) or combination 14 (expressing SoP450_1944 and SoP450_6085). Likewise, strain 17, expressing C-28 oxidase SoP450_1944 together with both C-23 oxidases (SoP450_2010 and SoP450_6085) yielded the same metabolites as strain 7 carrying SoP450_1944 and SoP450_2010 only. However, differently from strain 7 that yielded gypsogenic acid, in strain 17 unexpectedly only gypsogenin was detected thus demonstrating that concomitant expression of both C-23 oxidases would promote that oxidation of the C-23 position stops at the aldehyde moiety (gypsogenin) reducing the production of the more oxidized carboxy group (gypsogenic acid).

Production of Quillaic Acid in yeast requires an unusual combination of C-28/16 and two C-23 oxidases

The most unexpected result was obtained by metabolite profiling of strain 19 where the promiscuous C- 28/C-16 oxidase (SoP450_1049) was co-expressed with both C-23 oxidases (SoP450_2010 and SoP450_6085). This strain, beside all the products derived from C-28 and C-16 oxidation of p-amyrin (such as erythrodiol, oleanolic and echinocystic acid), was also found to synthesize 16-hydroxy- gypsogenic acid (similarly to strain 10) but differently it also accumulated quillaic acid and traces of gypsogenic acid (Fig. 15). This is to our knowledge not only the first time that quillaic acid has been heterologously produced in yeast, but it additionally also involves an unusual combination of enzymes with partially overlapping functions. Surprisingly, the quillaic acid pathway seems to require two C-23 oxidases that feed on each other products. For instance with our experiments we demonstrated that the first enzyme SoP450_1049 is sufficient for production of echinocystic acid (the C-28/C-16 oxidized form of -amyrin). Afterwards, the first C-23 oxidase (SoP450_2010) is capable of oxidizing the C-23 position into an alcohol moiety producing caulophyllogenin that is readily converted by the second C-23 enzyme (SoP450_6085) into quillaic acid (Fig. 15). As a further confirmation, the metabolite extract from yeast strain 19 was additionally analyzed by UPLC-MS analysis using a high resolution protocol that allows to distinguish more clearly between quillaic acid and gypsogenic acid (Fig. 15, second panel), confirming that the peak produced by yeast strain 19 unambiguously matches the quillaic acid standard. Example 4: Overexpression of newly discovered enzymes in transgenic S. officinalis hairy root lines

For this example, we generated different constructs for ectopic overexpression of the candidate genes in planta, more specifically in transformed hairy roots of S. officinalis, and assess the effect thereof on the production levels of a specific oleanane type saponin, named sapofectosid (i.e. also designated as SO1861) [11], Transgenic S. officinalis hairy root lines were generated via Agrobacterium rhizogenes infection of S. officinalis adult leaves adapting a previously described protocol [12], S. officinalis hairy roots were grown for two months on liquid Murashige and Skoog medium including vitamins supplied with 1.5 % sucrose. The hairy roots were harvested and ground in liquid nitrogen into a fine powder. Total metabolite extraction and subsequent LC-MS analysis was conducted as previously reported [13],

The transgenes selected for ectopic overexpression in S. officinalis included three of the newly discovered S. officinalis genes involved in triterpene production, namely SoBAS_1421, SoP450_1049 and SoUGT_2488. The coding sequence of each gene was cloned into the Gateway-compatible binary vector pK7WG2D [8], For each transgene four independent hairy root lines were cultured, analyzed and compared with four independent control lines (ectopically expressing the metabolically non-active green fluorescent protein GFP).

LC-MS profiling data of metabolite extracts from control hairy roots, expressing only GFP, was compared to those of hairy roots expressing the transgenes of interest. This analysis revealed that overexpression of either SoBAS_1421 or SoP450_1049 caused a substantial increase in SO1861 levels (Fig. 16). Conversely, overexpression of SoUGT_2488, encoding the putative UGT that catalyzes the transfer of glucuronic acid to the C-3 position of quillaic acid did not affect the SO1861 levels. Together these data illustrate that (i) overexpression of a single pathway enzyme can promote SO1861 production, thus demonstrating the utility of single chimeric genes to engineer S. officinalis saponin biosynthesis their and (ii) that not all of the committed pathway steps are rate-limiting, thus demonstrating that the utility of a particular single chimeric gene as an engineering tool is unpredictable. A schematic representation of the biosynthetic pathway of quillaic acid in S.officinalis is reported in figure 17.

Example 5: Overexpression of newly discovered enzymes in transgenic Gypsophila elegans hairy root lines

For this example, diverse constructs were generated to achieve the ectopic overexpression of the candidate genes in planta, specifically within the transformed hairy roots of Gypsophila elegans, and to evaluate their impact on the production levels of particular oleanane type saponins sharing structural and biochemical features with the designated SO1861. Transgenic G.elegans hairy root lines were established using Agrobacterium rhizogenes infection of G.elegans seedlings, following a previously established protocol (Pollier J. et al (2019) Plant J. 99:637- 654). These G. elegans hairy roots were cultivated for a span of two months in liquid Murashige and Skoog medium supplemented with vitamins and 1.5% sucrose. Afterward, the hairy roots were collected, frozen in liquid nitrogen, and pulverized into fine powder. The comprehensive extraction of metabolites and subsequent LC-MS analysis were carried out in accordance with previously documented procedures (Mertens J. et al (2016) Plant Physiol. 170:194-210).

The chosen transgenes for ectopic overexpression in G. elegans included each of the newly identified S. officinalis P450 genes linked to triterpene production. This involved both individual (namely SoP450_1944, SoP450_6085, SoP450_2010, SoP450_1049) and collective overexpression in a binary vector equipped with seven distinct transcriptional cassettes. These cassettes encompassed the aforementioned P450s as well as SoBAS_1421, SoSQE2, and eGFP, serving as a visual transformation marker. For this purpose, the Golden Gibson assembly method was employed for binary vector construction, enabling the incorporation of multiple transcriptional cassettes, as previously described (Aesaert S et al. 2022) Front. Plant Sci 13:883847). For the expression of individual transgenes, the Gateway-compatible binary vector pK7WG2D was utilized, as outlined in Example 4 for S. officinalis hairy roots . For each vector, three separate hairy root lines were cultivated, analyzed, and compared against three distinct control lines (expressing solely the metabolically inactive green fluorescent protein, GFP).

LC-MS profiling data of metabolite extracts from control hairy roots, expressing only GFP, was compared to those of hairy roots expressing the transgenes of interest. The effect of transgene expression was evaluated on the content of seven different saponins that were reported as representative of Gypsophila triterpenoid profile (Voutquenne-Nazabadioko L et al (2013) Phytochemistry 90:114-127) sharing substantial structural similarities with Sapofectosid. The outcome of this analysis revealed that the sole overexpression of SoP450_1049 (as illustrated in Fig. 18 - Gl_1049) or the concurrent expression of SoSQE2, SoBAS_1421, and all four Saponaria P450 genes as disclosed in this application (Fig. 18 - Gl_ALL_P450s) yielded a substantial increase in the production of a particular saponin, specifically C4_1567.681 (see Fig. 18). The latter saponin is a closely related structure to sapofectosid. While the simultaneous expression of multiple saponin pathway genes in the G. elegans hairy root line Gl_ALL_P450s appeared to enhance the production of C4_1567.681, the variations observed in comparison to hairy root lines expressing solely SoP450_1049 did not demonstrate statistical significance. Thus, the overexpression of a single gene in Gypsophila elegans is sufficient to increase the production of the sapofectosid homologue. Sequence listing

SEQ ID NO: 1 - S. officinalis P-amyrin synthase, SoBAS_1421 coding sequence (2283 bps)

SEQ ID NO: 2 - S. officinalis SoBAS_1421 translated nucleotide sequence (760 AA)

SEQ ID NO: 3 - S. officinalis SoP450_1944, C-28 oxidase coding sequence(1458 bps)

SEQ ID NO: 4 - S. officinalis SoP450_1944 translated nucleotide sequence(485 AA)

SEQ ID NO: 5 - S. officinalis SoP450_2010, C-23 oxidase coding sequence (1568 bps)

SEQ ID NO: 6 - S. officinalis SoP450_2010, C-23 oxidase translated nucleotide sequence(522 AA)

SEQ ID NO: 7 - S. officinalis SoP450_1049, C-28/C-16 oxidase coding sequence (1509 bps)

SEQ ID NO: 8 - S. officinalis SoP450_1049, C-28/C-16 oxidase translated nucleotide sequence (502 AA)

SEQ ID NO: 9 - S. officinalis SoP450_6085, C-23 oxidase coding sequence (1374 bps)

SEQ ID NO: 10 - S. officinalis SoP450_6085, C-23 translated nucleotide sequence (457 AA)

SEQ ID NO: 11 - Glycyrrhiza glabra GgBAS, -amyrin synthase coding sequence (2298 bps)

SEQ ID NO: 12 - Glycyrrhiza glabra GgBAS, p-amyrin synthase translate nucleotide sequence (765 AA)

SEQ ID NO: 13 - Medicago truncatula MTtHMGRl, (truncated HMG-CoA reductase MTR_5g026500) (1374 bps)

SEQ ID NO: 14 - Medicago truncatula MtHMGRl (truncated HMG-CoA reductase MTR_5g026500) (457 AA)

SEQ ID NO: 15 - Medicago truncatula MtMTRl, NADPH-cytochrome P450 reductase (Medtr3gl00160) (2079 bps)

SEQ ID NO: 16 - Medicago truncatula MtMTRl, NADPH-cytochrome P450 reductase (Medtr3gl00160) (692 AA)

SEQ ID NO: 17 Saponaria officinalis SoUGT_2488, Nucleotide-diphospho-sugar transferases - Cellulose synthase (2097 bps)

SEQ ID NO: 18 Saponaria officinalis SoUGT_2488, Nucleotide-diphospho-sugar transferases - Cellulose synthase (698 AA)

SEQ ID NO: 19 Saponaria officinalis SotHMGR, hydroxymethylglutaryl-CoA reductase (NADPH) (1443 bps)

SEQ ID NO: 20 Saponaria officinalis SotHMGR, hydroxymethylglutaryl-CoA reductase (480 AA)

SEQ ID 21: SoSQE2 Saponaria officinalis, Squalene Epoxidase 2 coding sequence (1557 bps)

SEQ ID 22: SoSQE2 Saponaria officinalis, Squalene Epoxidase 2 translated nucleotide sequence (518 AA) References

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Claims

Claims

1. A eukaryotic cell comprising at least one of the following chimeric genes: a. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 2 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 2 and transcription termination and polyadenylation signals, b. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 6 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 6 and transcription termination and polyadenylation signals, c. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 8 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 8 and transcription termination and polyadenylation signals, d. a promoter operably linked to a nucleotide sequence encoding SEQ ID NO: 10 or a nucleotide sequence encoding a sequence with 95% identity over the total length of SEQ ID NO: 10 and transcription termination and polyadenylation signals.

2. A eukaryotic cell according to claim 1 further comprising a chimeric gene encoding a feedbackinsensitive 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3. A eukaryotic cell according to claims 1 or 2 further comprising a chimeric gene encoding a NADPH-cytochrome P450 reductase.

4. A eukaryotic cell according to claim 1 which is a yeast cell and wherein said yeast cell comprises the chimeric genes a), b), c) and d) as cited in claim 1.

5. A eukaryotic cell according to claim 2 which is a yeast cell and wherein said yeast cell comprises the chimeric genes a), b), c) and d) as cited in claim 1.

6. A eukaryotic cell according to claim 3 which is a yeast cell and wherein said yeast cell comprises the chimeric genes a), b), c) and d) as cited in claim 1.

7. A eukaryotic cell according to claims 1 or 2 which is a plant cell.

8. A eukaryotic cell according to claim 7 which is a plant cell from the genus Saponaria or Gypsophila.

9. A plant cell from the genus Saponaria or Gypsophila which comprises a chimeric gene selected from a) or b) as cited in claim 1.

10. A method to produce quillaic acid in yeast comprising introducing the chimeric genes a), b), c) and d) as cited in claim 1 in said yeast, fermenting said transformed yeast and isolating quillaic acid from said fermented yeast.

11. A method according to claim 10 wherein the yeast cell further comprises a chimeric gene encoding a feedback-insensitive 3-hydroxy-3-methylglutaryl coenzyme A reductase. A method according to claim 11 wherein the yeast cell further comprises a chimeric gene encoding a NADPH-cytochrome P450 reductase. A method to produce sapofectosid in a Saponaria or Gypsophila species comprising introducing the chimeric gene a) or b) as cited in claim 1 into hairy roots of a Saponaria or Gypsophila species, growing said transformed hairy roots and isolating sapofectosid from said grown transformed hairy roots.