WO2025168705A1 - Means and methods for the production of saponins with endosomal escape-enhancing properties - Google Patents

Abstract

This invention provides methods for the production of glycosylated triterpenoids which function as endosomal escape enhancers. Specifically, the invention provides UDP glycosyltransferases (UGTs) useful for the biosynthesis of these glycosylated triterpenoid saponins. The invention also provides in vitro systems and plant extracts comprising UDP glycosyltransferases and sapofectosid precursors.

Description

MEANS AND METHODS FOR THE PRODUCTION OF SAPONINS WITH ENDOSOMAL ESCAPEENHANCING PROPERTIES

Field of the invention

This invention relates to the field of secondary metabolite production in plants, particularly triterpenoids, more particularly glycosylated triterpenoids which function as endosomal escape enhancers, even more particularly to UDP glycosyltransferases (UGTs) useful for the biosynthesis of glycosylated triterpenoid saponins which are endosomal escape enhancers.

Introduction to the invention

In the context of modern therapeutics, where targeted protein- and DNA-based treatments predominantly enter cells via the endocytic route enclosed in endosomal vesicles, a significant challenge lies in ensuring effective drug delivery to the cytosol for optimal functionality. It has been discovered that specific highly glycosylated triterpenoids from Saponaria officinalis possess the unique ability to destabilize endosomal membranes, allowing drugs to "escape" from vesicles into the cytosol, thereby significantly enhancing treatment efficacy. These complex triterpenoid saponins, referred to as Endosomal Escape Enhancers (EEEs), exist at very low levels in plants and comprise a complex isomeric mixture. The biosynthesis of these compounds involves various cellular compartments and classes of enzymes. Cyclase enzymes cyclize linear sterol into a beta amyrin pentacyclic backbone, while membrane-bound P450s selectively oxidize positions of the carbon backbone. This allows UDP glycosyltransferase enzymes to decorate the triterpenoid scaffold with diverse sugar moieties (D- glucose, D-galactose, D-glucuronic acid, D-galacturonic acid, L-rhamnose, L-arabinose, D-xylose, and D- fucose), imparting amphipathicity and bioactive properties to the otherwise mostly apolar backbone. These intricate metabolic networks govern the production of EEE molecules, such as Sapofectosid, posing challenges for their synthetic production.

Sapofectosid (herein further designated as SO1861), acknowledged for its roles as a transfection reagent and a potent endosomal escape agent, was recently identified in the medicinal plant Saponaria officinalis, predominantly in perennial parts like roots and woody stems. Notably, Sapofectosid is not the most prevalent saponin in Saponaria; conversely, other saponins with structural features similar to sapofectosid, like for example SO1700, are ubiquitously distributed in Saponaria tissues and likely serve as intermediate or late-pathway precursors to Sapofectosid (SO1861).

Recognizing the time and labor-intensive nature of metabolic engineering in non-model plant species, which necessitates the establishment of transformation protocols from scratch, this invention addresses this challenge by identifying sugar transferases capable of converting in-vitro SO1700 into sapofectosid through the addition of glucose moieties in-vitro. The present invention provides UGT enzymes capable of finalizing the conversion of major saponins into Sapofectosid. In particular, we have identified two UDP-glycosyltransferase (UGT) enzymes in S. officinalis. The first enzyme, SoUGT_2207 (depicted in SEQ ID NO: 2), demonstrates the capability to attach a glucose moiety to SO1700, resulting in the formation of Sapofectosid. Moreover, it can glycosylate a saponin structurally related to SO1700, named SO1730 (see Fig. 4), yielding a compound akin to Sapofectosid, herein referred to as SO1891. The second UGT, named SoUGT_10304 (depicted in SEQ ID NO: 4), exhibits proficiency in adding either UDP-Glucose or UDP-Xylose to the SQ1700 precursor. This enzymatic activity leads to the production of Sapofectosid (SO1861) and a related saponin known as Saponarioside A (herein referred to also as SO1831), where one glucose molecule is replaced by xylose.

Our invention offers a solution to the challenges associated with chromatographic techniques for the separation and extraction of SO1861 from complex plant biomass. These challenges arise from the presence of numerous compounds sharing similar mass and chemical features. Furthermore, this invention has significant implications for increasing the overall yield and sustainability of SO1861 extraction. Traditional methods currently rely on the extraction of SO1861 from plant roots, necessitating the destruction of the perennial root apparatus of Saponaria officinalis plants. In contrast, our method enables a cyclic harvest of aerial plant parts for the extraction of total metabolites and the in-vitro conversion of late-pathway intermediates into SO1861. Importantly, this approach leaves the roots untouched, allowing them to sprout again and produce new aerial parts for subsequent harvests and conversions.

Legends to the figures

Figure 1: Expression trends for selected transcripts. The left and right panels report data as transcripts abundance (Counts per Million) and expression trends as scaled expression values respectively.

Figure 2: Agarose gel electrophoresis showing DNA bands corresponding to amplified target genes from cDNA. Target gene names are reported within the grey boxed below the gel bands. Codes report the cDNA used in PCR reaction: J = Jasmonate, 6 = six hours after treatment, L = leaves, P = plantlets.

Figure 3: SDS-PAGE analysis of cell-free produced and purified SoUGT_10304 and SoUGT_2207 from S. officinalis, respectively. The enzymes were produced in BYL and purified via a C-terminal Strep-tag. After dialysis, protein concentrations were determined photometrically, and 1000 ng (SoUGT_10304) and 800 ng (SoUGT_2207) of each protein were analyzed on SDS-PAGE alongside enhanced yellow fluorescent protein (eYFP) standards in four different amounts. The gels were stained with Coomassie, and the standards were used for densitometric concentration evaluation. Figure 4: Inferred chemical structures of main precursors and products: A) SO1700, B) SO1730, C) SO1861 (aka Sapofectosid), D) SO1831 (aka Saponarioside A), E) SO1891.

Figure 5: Extracted Ion Chromatograms of SO1861 derived from HPLC analysis of in-vitro reactions of candidate UGTs incubated with SO1700 and UDP-GIc or UDP-Xylose. Only reactions that included one of the two UGTs (highlighted by green and blue arrow) in presence of SO1700 and UDP-GIc produced the target compound. Reaction containing only UDP-Xyl as only cofactor or No Enzyme controls (yellow and red chromatograms), didn't yield any SO1861.

Figure 6: Extracted Ion Chromatograms of SO1831 derived from HPLC analysis of in-vitro reactions of candidate UGTs incubated with SO1700 and UDP-GIc or UDP-Xylose. Only reactions that included SoUGT_10304 (highlighted by purple arrow) in presence of SO1700 and UDP-Xyl yielded SO1831. Reaction containing only UDP-GIc as only cofactor or No Enzyme controls (green and red chromatograms), didn't produce any SO1831.

Figure 7: Extracted Ion Chromatograms of SO1891 derived from HPLC analysis of in-vitro reactions of candidate UGTs incubated with SO1730 and UDP-GIc or UDP-Xylose. Only reactions that included SoUGT_2207 (highlighted by grey arrow) in presence of SO1730 and UDP-GIc yielded SO1891. Reaction containing only UDP-GIc as only cofactor or No Enzyme controls (green and red chromatograms), didn't produce any SO1891.

Figure 8: Detection of substrate (left panel) and product (right panel) abundances as signal intensities by HPLC analysis of in-vitro reactions. A) Analysis of substrate abundances revealed pointed to a conversion of about 12% and 21% of SO1700 into SO1861 by SoUGT_2207 and SoUGT_10304 respectively (SO1861 levels are displayed as blue bars in panel B). B) SO1700 conversion reached 83% by SoUGT_10304 using UDP-Xyl, thus producing SO1831 (orange bar).

Figure 9: Detection of SO1891 product abundance as signal intensities by HPLC analysis of in-vitro reactions. Only reactions that included SoUGT_2207 (highlighted by grey arrow) in presence of SO1730 and UDP-GIc yielded SO1891. SO1891 was produced as minor secondary product therefore estimation of substrate consumption was not possible.

Figure 10: Luminescence-based estimation of Michaelis-Menten curves for each enzyme by UDP-Glo™ assay kit.

Figure 11: Schematic summary of enzymatic reactions carried by SoUGT_2207 and SoUGT_10304 presented in this work. Figure 12: HPLC analysis of SO1861 (top left) standard as well as SO1700 (bottom left) and SO1730 (bottom right) precursors. Green circles highlight the exact masses detected by MS analysis while red circles highlight MS/MS fragment (m/z 955 Da) that is specific to EEE molecules sharing same structural features as SO1861. On the top right corner is reported the structure of SO1861 while in red is highlighted the part corresponding to the m/z 955 diagnostic mass resulting from MS/MS fragmentation.

Figure 13: LC-MS relative abundance of sapofectosid (SO1861 - green bars) and related compound SO1831 (pink bar) in in-vitro reactions including S.officinalis leaves extract (YL) with SoUGT_10304 (UGT_10304) and SoUGT_2207 (UGT_2207) in presence of 100 uM UDP-glucose (UDP-GIc) or UDP-xylose (UDP-xylose). In accordance with previous examples, SoUGT_10304 displays higher plasticity for both products, SO1831 and SO1861, and substrates, UDP-GIc and UDP-Xyl. Differently SoUGT_2207 accepts only UDP-GIc as sugar donor, producing only SO1861 in higher abundance as compared to SOUGT_10304.

Figure 14: LC-MS detected levels of sapofectosid (SO1861) and related compound SO1831, in metabolite extracts from transgenic Gypsophila elegans hairy roots overexpressing Saponaria officinalis genes p- amyrin synthase (SoBAS, pink), SoUGT_2207 (green) or SoUGT10304 (orange). All transgenic hairy root lines display comparable levels of SO1831 while only hairy root lines overexpressing SoUGT_2207 show a substantial increase in SO1861 production.

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 UDP-glycosyltransferase biosynthetic enzymes from Saponaria officinalis, which enzymes can be used for the in vitro conversion of late pathway precursors of sapofectosid. Sapofectosid has a molecular mass of about 1861 Dalton (herein abbreviated as SO1861). The term "about" is used in the claims because depending on the ionization mode of the mass spectrometer to measure sapofectosid the mass slightly varies. For example, the neutral mass of sapofectosid is 1861 Dalton, but if the ionization yields the negative ion then the mass is 1860 Dalton. Late pathway precursors of sapofectosid have molecular masses of about 1730 (abbreviated herein as SO1730) and of molecular mass about 1700 (abbreviated herein as SO1700).

In one embodiment the invention provides a nucleotide sequence encoding a protein having at least 70% amino acid identity over the total length of SEQ ID NO: 2. SEQ ID NO: 2 is a UDP-glucosyltransferase. This enzyme transfers glucose from UDP-glucose to the C28 position of the carbon backbone of SO1700 or SO1730. SEQ ID NO: 2 is not present in the public domain and its closest homologue is an absicisate beta-glucosyltransferase from Camellia lanceoleosa (44% amino acid identity with SEQ ID NO: 2).

Accordingly, the invention provides a protein sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity over the total length of SEQ ID NO: 2.

In another embodiment the invention provides a nucleotide sequence encoding a protein having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity over the total length of SEQ ID NO: 2.

In yet another embodiment the invention provides a cDNA sequence encoding a protein having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity over the total length of SEQ ID NO: 2.

The cDNA sequence encoding SEQ ID NO: 2 is depicted in SEQ ID NO: 1.

The instant invention also provides a second UDP-glycosyltransferase enzyme which amino acid sequence is depicted in SEQ ID NO: 4. SEQ ID NO: 4 has combined UDP-glucosyltransferase activity and UDP-xylosyltransferase activity. SEQ ID NO: 4 is disclosed in W02024003012.

The enzyme having at least 70%, at least 80%, at least 85%, at least 90%, at least 95% amino acid identity over the total length of SEQ ID NO: 2 and the enzyme having at least 70%, at least 80%, at least 85%, at least 90%, at least 95% amino acid identity over the total length of SEQ ID NO: 4 can be used in an in vitro system to modify the late pathway precursors of sapofectosid, SO1700 and/or SO1730, into compounds with endosomal escape-enhancing properties, such as for example SO1861 (sapofectosid). SEQ ID NO: 2 and SEQ ID NO: 4 can be made recombinantly, can be isolated from a plant cell extract or can be made synthetically. Preferably, SEQ ID NO:2 and SEQ ID NO: 4 are purified enzymes. The concentration of typical enzymes and substrates in a cell are typically in the low pM range (see Kathy R. Able et al (1990) J. Theor. Biol. 143, 163-195 - Table 2).

In a particular embodiment the invention provides an in vitro system comprising: a. between I mM to 100 mM of an enzyme having at least 70%, at least 80%, at least 90%, at least 95% amino acid identity over the total length of SEQ ID NO: 2, b. and/or including 1 mM and 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4, c. lOmM to 500 mM of UDP-glucose, and d. sapofectosid precursors SO1700 and/or SO1730.

In another particular embodiment the invention provides an in vitro system comprising: a. between 10 mM to 50 mM of an enzyme having at least 70%, at least 80%, at least 90%, at least 95% amino acid identity over the total length of SEQ ID NO: 2, b. and/or including 10 mM and 50 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4, c. lOOmM to 500 mM of UDP-glucose, and d. sapofectosid precursors SQ1700 and/or SQ1730.

An in vitro system is equivalent to a buffer and is an environment with an optimal pH for the activity of the enzymes SEQ ID NO: 2 and/or SEQ ID NO: 4 including ions and salts similar to the osmolality of a eukaryotic cytoplasm.

In another embodiment the invention provides a plant extract comprising: a. between 1 mM to 100 mM of an enzyme having at least 70%, at least 80%, at least 90%, at least 95% amino acid identity over the total length of SEQ ID NO: 2, b. and/or including 1 mM and 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4, c. lOmM to 500 mM of UDP-glucose.

In another particular embodiment the invention provides a plant extract comprising: a. between 10 mM to 50 mM of an enzyme having at least 70%, at least 80%, at least 90%, at least 95% amino acid identity over the total length of SEQ ID NO: 2, b. and/or including 10 mM and 50 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4, and c. lOOmM to 500 mM of UDP-glucose.

In another particular embodiment the plant extract further comprises sapofectosid precursors SQ1700 and/or SQ1730. In a particular embodiment the plant extract is prepared from a plant selected from the family of Caryophyllaceae plants.

In another particular embodiment the plant is selected from the genus Saponaria, Gypsophila or Agrostemma.

In yet another particular embodiment the plant extract is prepared from a plant from the genus Quillaja.

In yet another embodiment the invention provides a method to produce sapofectosid comprising adding to an in vitro system between 1 mM to 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, optionally also including 1 mM and 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: A, lOmM to 500 mM of UDP-glucose and sapofectosid precursors SQ1700 and/or SO1730.

In yet another embodiment the invention provides a method to produce sapofectosid comprising adding to an in vitro system between 10 mM to 50 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, optionally also including 10 mM and 50 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: A, 100 mM to 500 mM of UDP- glucose and sapofectosid precursors SQ1700 and/or SQ1730.

In yet another embodiment the invention provides a method to produce sapofectosid comprising adding to a plant extract between 1 mM to 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, and/or including 1 mM to 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4 and 10 mM to 500 mM of UDP-glucose.

In yet another embodiment the invention provides a method to produce sapofectosid comprising adding to a plant extract between 10 mM to 50 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, and/or including 10 mM to 50 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4 and between 100 mM to 500 mM of UDP- glucose.

In a specific embodiment in the method sapofectosid precursors SQ1700 and/or SQ1730 are added.

In specific embodiments in the method the plant is selected from the genus Saponaria, Gypsophila, Agrostemma or Quillaja.

In yet another embodiment the invention provides a Saponaria or Gypsophila plant, plant cell or hairy root having a loss of function in the gene encoding for an amino acid having at least 70% amino acid identity over the total length of SEQ ID NO: 4 and said plant, plant cell or hairy root comprises a chimeric gene encoding for a protein having at least 70% amino acid identity over the total length of SEQ ID NO: 2.

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 cells, b) a nucleotide sequence encoding an amino acid sequence with at least 70% identity over the total length of SEQ ID NO: 2 and c) a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.

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 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.

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.

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. The term 'a loss of function-allele' can be obtained by methods well-known in the art. In certain embodiments, the methods can comprise making a deletion, an insertion and/or a substitution which results in a loss-of-function allele of the endogenous crop gene. Gene editing molecules of use in methods provided herein include molecules capable of introducing a double-strand break ("DSB") or single-strand break ("SSB") at a specific site or sequence in a double-stranded DNA, such as in genomic DNA or in a target gene located within the genomic DNA as well as accompanying guide RNA. In certain embodiments, the loss-of-function allele results from introduction of a DSB at a target site in the endogenous crop gene to induce non-homologous end joining (NHEJ) at the site of the break followed by recovery of desired loss-of-function alleles. In certain embodiments, the loss-of-function allele results from introduction of a DSB at a target site in the endogenous crop gene followed by homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), or NHEJ to introduce a desired donor or other DNA template polynucleotide at the DSB, followed by recovery of the desired loss-of-function allele. Examples of such gene editing molecules include: (a) a nuclease comprising an RNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNA endonuclease (RdDe), a class 1 CRISPR type nuclease system, a type II Cas nuclease, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Casl2a nuclease, a nCasl2a nickase, a Casl2d (CasY), a Casl2e (CasX), a Casl2b (C2cl), a Casl2c (C2c3), a Casl2i, a Casl2j, a Casl4, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) or nickase, a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or engineered meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effectuating site-specific alteration (including introduction of a DSB or SSB) of a target nucleotide sequence; (c) a guide RNA (gRNA) for use with an RNA-guided nuclease, or a DNA encoding a gRNA for use with an RNA-guided nuclease; (d) optionally donor DNA template polynucleotides suitable for insertion at a break in genomic DNA by homology-directed repair (HDR) or microhomology-mediated end joining (MMEJ); and (e) optionally other DNA templates (e.g., dsDNA, ssDNA, or combinations thereof) suitable for insertion at a break in genomic DNA (e.g., by non- homologous end joining (NHEJ). In certain embodiments, the loss-of-function allele of the endogenous crop gene and plant cells, parts including seeds, and plants comprising the loss-of-function allele of the endogenous crop gene are generated by CRISPR technology. CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016/0138008A1 and US2015/0344912A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 Al. Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in published WO 2015/131101. Methods of using CRISPR technology for genome editing in plants are disclosed in US Patent Application Publications US 2015/0082478A1 and US 2015/0059010A1 and in published WO 2016/007347. In certain embodiments, an RNA-guided endonuclease that leaves a blunt end following cleavage of the target site is used. Blunt- end cutting RNA-guided endonucleases include Cas9, Casl2c, Casl2i, and Cas 12h (Yan et al., 2019). In certain embodiments, an RNA-guided endonuclease that leaves a staggered single stranded DNA overhanging end following cleavage of the target site following cleavage of the target site is used. Staggered-end cutting RNA-guided endonucleases include Casl2a, Casl2b, and Casl2e.

Guide RNA molecules comprising a spacer RNA molecule which targets the endogenous crop genes or an allelic variant thereof are provided. CRISPR-type genome editing can be adapted for use in the plant cells and methods provided herein in several ways. CRISPR elements, e.g., gene editing molecules comprising CRISPR endonucleases and CRISPR guide RNAs including single guide RNAs or guide RNAs in combination with tracrRNAs or scoutRNA, or polynucleotides encoding the same, are useful in effectuating genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny. In certain embodiments, the CRISPR elements are provided directly to the eukaryotic cell (e.g., maize plant cells), systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of a cell free synthetic process (e.g., in vitro translation), or as isolated or semi-purified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, crop plants or crop plant cells used in the systems, methods, and compositions provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Cas9, a Cpfl-type or other CRISPR endonuclease). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Guide RNAs (sgRNAs or crRNAs and a tracrRNA) to form an RNA-guided endonuclease/guide RNA complex which can specifically bind sequences in the gDNA target site that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of RNA-guided endonuclease typically informs the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g., 5'-NGG are typically targeted for design of crRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'- NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), 5'- NNGRRT or 5'-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5'-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5'-TTN or 5'-TTTV, where "V" is A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with Casl2a proteins. In some instances, Casl2a can also recognize a 5'-CTA PAM motif. Other examples of potential Casl2a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 Al, which is incorporated herein by reference for its disclosure of DNA encoding Cpfl endonucleases and guide RNAs and PAM sites.

In certain embodiments, the loss-of-function allele of the endogenous crop gene and plant cells, parts including seeds, hairy roots obtained from plants and plants comprising the loss-of-function allele of the endogenous crop gene are generated by use of zinc finger nucleases or zinc finger nickases. Zinc-finger nucleases are site-specific endonucleases comprising two protein domains: a DNA-binding domain, comprising a plurality of individual zinc finger repeats that each recognize between 9 and 18 base pairs, and a DNA-cleavage domain that comprises a nuclease domain (typically Fokl). The cleavage domain dimerizes in order to cleave DNA; therefore, a pair of ZFNs are required to target non-palindromic target polynucleotides. In certain embodiments, zinc finger nuclease and zinc finger nickase design methods which have been described (Urnov et al. (2010) Nature Rev. Genet., 11:636 - 646; Mohanta et al. (2017) Genes vol. 8,12: 399; Ramirez et al. Nucleic Acids Res. (2012); 40(12): 5560-5568; Liu et al. (2013) Nature Communications, 4: 2565) can be adapted for use in the methods set forth herein. The zinc finger binding domains of the zinc finger nuclease or nickase provide specificity and can be engineered to specifically recognize any desired target DNA sequence. The zinc finger DNA binding domains are derived from the DNA-binding domain of a large class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains a tandem array of at least three zinc "fingers" each recognizing a specific triplet of DNA. A number of strategies can be used to design the binding specificity of the zinc finger binding domain. One approach, termed "modular assembly", relies on the functional autonomy of individual zinc fingers with DNA. In this approach, a given sequence is targeted by identifying zinc fingers for each component triplet in the sequence and linking them into a multifinger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to accommodate the ability of zinc fingers to contact neighboring fingers as well as nucleotide bases outside their target triplet. Typically, the engineered zinc finger DNA binding domain has a novel binding specificity, compared to a naturally occurring zinc finger protein. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of databases of triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, e.g., US Patents 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) can be adapted for use in the methods described herein. In addition, enhancement of binding specificity for zinc finger binding domains has been described in US Patent 6,794,136, incorporated herein by reference in its entirety. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. Examples of linker sequences are publicly known, e.g., see US Patents 6,479,626; 6,903,185; and 7,153,949, incorporated herein by reference in their entirety. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fokl. This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fokl as part of a ZFN requires two adjacent and independent binding events, which must occur in both the correct orientation and with appropriate spacing to permit dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites. Fokl variants with enhanced activities have been described and can be adapted for use in the methods described herein; see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96 - 107.

In certain embodiments, the loss-of-function allele of the endogenous crop gene and plant cells, parts including seeds, and plants comprising the loss-of-function allele of the endogenous crop gene are generated by use of TAL-effector nucleases or TALENs. Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to a non-specific DNA cleavage domain to prepare genome editing proteins, referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl, can be conveniently used. Methods for use of TALENs in plants have been described and can be adapted for use in the methods described herein, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA, 108:2623 - 2628; Mahfouz (2011) GM Crops, 2:99 - 103; and Mohanta et al. (2017) Genes vol. 8,12: 399). TALE nickases have also been described and can be adapted for use in methods described herein (Wu et al.; Biochem Biophys Res Commun. (2014);446(l):261-6; Luo et al; Scientific Reports 6, Article number: 20657 (2016)). Various treatments can be used for delivery of gene editing molecules and/or other molecules to a plant cell. In certain embodiments, one or more treatments is employed to deliver the gene editing or other molecules (e.g., comprising a polynucleotide, polypeptide or combination thereof) into a plant cell, e.g., through barriers such as a cell wall, a plasma membrane, a nuclear envelope, and/or other lipid bilayer. In certain embodiments, a polynucleotide-, polypeptide-, or RNP (ribonucleoprotein) -containing composition comprising the molecules are delivered directly, for example by direct contact of the composition with a plant cell. Aforementioned compositions can be provided in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant, plant part, plant cell, or plant explant (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a plant cell or plant protoplast is soaked in a liquid genome editing molecule-containing composition. In certain embodiments, the composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In certain embodiments, the composition is introduced into a plant cell or plant protoplast, e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in US Published Patent Application 2014/0287509, incorporated by reference in its entirety herein. Other techniques useful for delivering the composition to a eukaryotic cell, plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In certain embodiments, the composition is provided by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the genome editing molecules (e.g., RNA dependent DNA endonuclease, RNA dependent DNA binding protein, RNA dependent nickase, ABE, or CBE, and/or guide RNA); see, e.g., Broothaerts et al. (2005) Nature, 433:629 - 633). Any of these techniques or a combination thereof are alternatively employed on a plant explant, plant part or tissue or intact plant (or seed) from which a plant cell is optionally subsequently obtained or isolated; in certain embodiments, the composition is delivered in a separate step after the plant cell has been isolated.

In certain embodiments, the population of crop plant cells, parts, or plants which are screened for the presence of a loss-of-function allele in the endogenous crop gene have been subjected to one or more mutagenesis treatments. Loss-of-function alleles of the endogenous crop gene can be generated by mutagenesis methods known in the art, such as chemical mutagenesis or radiation mutagenesis. Suitable chemical mutagens include ethyl methanesulfonate (EMS), sodium azide, methylnitrosourea (MNU), and diepoxybutane (DEB). Suitable radiation includes x-rays, fast neutron radiation, and gamma radiation.

Crop plant cells, parts, or plants comprising a loss-of-function allele of the endogenous crop gene can be generated using mutagenesis and identified by TILLING (Targeting Induced Local Lesions IN Genomes) or identified using EcoTILLING. TILLING is a general reverse genetics technique that uses mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high throughput screens for the discovery of mutations. In addition to allowing efficient detection of induced mutations, high-throughput TILLING technology is ideal for the detection of natural mutations. EcoTILLING is a method that uses TILLING techniques to look for natural mutations in individuals (Barkley and Wang. Current genomics vol. 9,4 (2008): 212-26. doi:10.2174/138920208784533656). Identified mutations can then be introduced into desirable genetic backgrounds by crossing the mutant with a plant of the desired genetic background and performing a suitable number of backcrosses to cross out the originally undesired parent background. A more detailed description of methods and compositions for TILLING are disclosed in US Patent Application Publication 2004/0053236 Al, which is incorporated herein by reference in its entirety and can be adapted for use in the methods provided herein for identifying crop plant cells, parts, or plants comprising a loss-of-function allele of the endogenous crop gene.

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

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 conversion by glycosylation of Saponaria officinalis high-molecular-weight saponins into Sapofectosid

To identify UDP-Glycosyltransferases involved in late glycosylation steps of the endosomal escape enhancer saponin (Sapofectosid™) Saponaria officinalis, co-expression analysis and homology sequence comparison were conducted on transcriptome datasets. 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.jelitto.com/, Schwarmstedt, Germany) (Fig 1).

RNA sequencing datasets were used to draft an S. officinalis reference transcriptome, and RNA extracted from the sampled tissues was single-end sequenced via Illumina HiSeq 6000 with single-end read lengths of 100 bp for gene expression profiling. Additionally, RNA was used as a template for cDNA synthesis, and the draft transcriptome was produced using public datasets available through the 1KP project (7). Transcripts levels were estimated by mapping single-end reads on the S. officinalis transcriptome using Salmon (8) 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 (12) 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. l.l SoUGT 2207

The first UGT enzyme (hereafter referred to as SoUGT_2207, see SEQ ID NO: 1 and 2) showed coexpression with other genes previously characterized for encoding enzymes participating in the early steps of triterpenoid saponin biosynthesis in S. officinalis. The SoUGT_2207 full coding sequence and predicted protein are reported as SEQ ID NOs: 1 and 2.

1.2 SOUGT 10304

The second UGT considered in this application (hereafter referred to as SoUGT_10304, see SEQ ID NO: 3 and 4) was not identified by co-expression analysis with previously characterized genes but instead by sequence homology showing substantial degree of identity with a Xylosyltransferase from Quillaja saponaria that is active on C28 position of quillaic acid (10) . The SoUGT_10304 full coding sequence and predicted protein are reported as SEQ ID NOs: 3 and 4. Example 2: Cloning candidate genes from S. officinalis and generation of expression constructs for heterologous protein production

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

Two PCR reactions were conducted for each gene, utilizing either leaf or root cDNA derived from plants that underwent elicitation with 50 pM MeJA for 6 hours as a template. The PCR reactions were prepared in a total volume of 25 pl using Q5 polymerase (New England Biolabs), following the manufacturer's instructions. For amplifying the coding sequences of the genes SoUGT_2207 and SoUGT_10304, the PCR thermal cycling comprised an initial denaturation at 98°C (30 sec), followed by 3 cycles of denaturation (98°C, 10 sec), annealing (60°C, 20 sec), extension (72°C, 1 min), and then 30 cycles using the same conditions but with an increase in the annealing temperature to 70°C, concluding with a final extension at 72°C (5 min).

Both genes were successfully amplified from both leaf and root cDNA samples. However, stronger bands for SoUGT_2207 (primers 07_F_GW and 07_R_GW) were observed in the root cDNA, whereas SoUGT_10304 amplification (primers 04_F_GW and 04_R_GW), although overall less efficient than the first gene, exhibited comparable results in both cDNAs (Fig. 2). Subsequently, the PCR product obtained from root amplification for each gene was purified and incorporated into the Gateway entry vector pDONR221 in accordance with the manufacturer's protocols. The resulting plasmids were introduced into E. coli DH5a, followed by extraction and sequencing to confirm the presence of correct inserts. Subsequent to verification, each gene within the entry clones served as a template for downstream applications, such as transferring into plasmid vectors compatible with expression in BYL-cell-free systems.

The coding sequences of the two candidate genes were subsequently separately transferred from pDNR221 gateway vectors into BYL-cell free compatible expression vector pLenEx as previously described (2) using PCR amplification and Gibson assembly and attaching the sequence coding for the Strep-tag to the 3'end of the genes; correct sequence insertion was confirmed by DNA Sanger sequencing. Larger amounts of the pLenEx vectors carrying each of the two UGTs, SoUGT_2207 and SoUGT_10304, necessary for cell-free expression were obtained using the NucleoBond Xtra Midi Plus kit (Macherey & Nagel, Duren, Germany).

Example 3: Production of two potential UDP-glycosyltransferases (UGTs) from S. officinalis in BYL cell- free lysate

The two potential UGTs from S. officinalis were produced by in-vitro transcription and translation using a cell-free lysate (BYL) derived from Nicotiana tabacum cv. BY-2 cells (3). BYL was prepared as described in Buntru et al. (2). The vector DNA was then used for coupled transcription-translation reactions in BYL as described before (1) with minor modifications: for the production of each UGT 3.9-4.9 ml BYL and 6.5- 9.5 pg vector DNA were used for production of SoUGT_2207 and SoUGT_10304, respectively. Transformation mixes were performed in 96 well plates (150 pl reaction per well) for two days. Example 4: Purification of two potential UGTs via Strep-tag affinity chromatography

For purification of the cell-free produced UGTs the BYL was centrifuged twice at 16,000xg for 5 min, transferring the supernatant to a fresh Eppendorf tube each time. The supernatant was then loaded onto a 1 ml Strep-Tactin®XT Superflow® high-capacity column (IBA Lifesciences, Gottingen, Germany) that had been equilibrated with lx washing buffer (100 mM Tris pH 8.0, 150 mM NaCI, 1 mM EDTA). The column was washed with 6 ml lx washing buffer and elution was performed with 3 ml BXT buffer (100 mM Tris pH 8.0, 150 mM NaCI, 1 mM EDTA, 50 mM biotin) of which the first 0.5 ml were discarded. The purified UGTs were dialyzed using a slide-A-Lyzer G2 cassette (cut-off 10 kDa, size 3 ml; Thermo Fisher Scientific, Waltham, MA, United States) (2x 2 I; overnight and 2 h at 4°C in cold room) or Vivaspin 6 centrifugal concentrators (cut-off 10000 MWCO; Sartorius, Gottingen, Germany) against 100 mM Tris pH 8.0, 150 mM NaCI. When the centrifugal concentrators were used, the purified UGTs were also concentrated in the same step.

The purified and dialyzed UGTs were analyzed by SDS-PAGE on precast NuPAGE 4-12% (w/v) polyacrylamide Bis-Tris gels (Thermo Fisher Scientific) alongside PageRuler Prestained Protein Ladder markers (Thermo Fisher Scientific). The gels were stained with Coomassie Brilliant Blue R-250. Determination of protein concentration was done photometrically at 280nm and densitometrically using the Coomassie stained SDS-PAGE (Fig. 3).

Example 5: In-vitro production of SO1861 from precursors SQ1700 and SO173Q by the isolated S. officinalis UGTs

For this example, we evaluated the capacity of our two candidate UGTs to glycosylate Sapofectosid late pathway intermediates that were named SO1700 and SO1730 by in-vitro glycosylation assays. These two compounds were identified and purified from S. officinalis biomass by EXTRASYNTHESE (https://www.extrasynthese.com/) as major saponins in leaves and roots of S. officinalis. According to HPLC analysis and MS/MS fragmentation pattern, SO1700 and SO1730 (Fig. 12) share with SO1861 the same structure differing from it only by the lack of one Glucose moiety (SO1700) or one Xylose (SO1730). This implies that addition of either one Glucose molecule onto SO1700 or one Xylose molecule onto SO1730 would yield SO1861.

The glycosyltransferase activity assay was performed in a final volume of 100 ul using purified proteins as described in previous section. Each reaction contained 20 mM Tris-HCI, 15 mM MgCI2 (pH = 7.5), 50 ug of UGT_2207 and 40 ug of UGT_10304 with the presence of 100 pM SO1700 or SO1730 and 100 pM UDP-Glucose or Xylose were incubated at 30 degrees overnight. Subsequently, the reaction mixtures were halted by adding methanol with 100 pM glycyrrhizin as an internal standard and centrifuged at 13500 rpm for 30 min for protein precipitation. Supernatant were therefore collected and sent for HPLC analysis at the Core Mass Spectrometry Facility of the University of Edinburgh (EdinOmics).

Our findings demonstrate that SoUGT_2207 exhibits specific activity towards UDP-GIc, enabling the glycosylation of both saponin substrates, SO1700 and SO1730. This results in the production of SO1861 (Sapofectosid) from SO1700 and SO1891 from SO1730, with SO1700 being the predominant substrate and SO1861 the main product. In case of the latter we estimated a conversion rate of SO1700 into SO1861 of 12%, in our conditions. Conversely, SoUGT_10304 specifically glycosylates SO1700 but can accept both UDP-Xylose and UDP-GIc. This leads to the predominant formation of SO1831 (estimated SO1700 conversion rate 83%) when utilizing UDP-Xylose and SO1861 (our target compound, Sapofectosid) when utilizing UDP-GIc (estimated SO1700 conversion rate 21%). Results are presented in Fig. 5 to 9.

Example 6: Determination of enzyme kinetics via UDP-Glo™ in-vitro luminescence based glycosylation assays

Briefly, UDP-Glo™ Glycosyltransferase Assays relies on the fact that upon enzymatic addition of UDP- sugar moiety by UGTs onto a compatible substrate (in our case a triterpenoid saponins SO1700 and SO1730), free UDP is released in the medium. Subsequently the kit's reagents convert UDP into ATP, which is utilized by the enzyme Luciferase (provided with the kit) to transform D-Luciferin into Oxyluciferin, generating light. The quantity of luminescence emitted during this process correlates directly with the amount of glycosylated products and it can be quantified using a luminescence detector (Promega GlowMax®). For each enzyme, SoUGT_2207 and SoUGT_10304, reactions were performed in triplicate and executed across a range of increasing saponin concentrations spanning from 0 to 400 pM depending on enzyme and substrates. The reactions were carried out in 25 ul at 30 degrees Celsius for 1 hour in 20 mM Tris-HCI, 15 mM MgCI2 (pH = 7.5), in the presence of 600 ng of purified enzyme, either SoUGT_2207 or SoUGT_10304, with 100 pM of either UDP-Glucose or UDP-Xylose. Protein concentrations for this assay were empirically determined to reduce background noise thus maximizing sensitivity. Different concentrations of SO1861 precursors, SO1700 or SO1730 were instead adopted to infer reaction kinetics as afore mentioned. Subsequently, the reactions were halted by adding UDP-Glo reagents and luminescence was recorded one hour later as specified by the manufacturer instructions.

Analysis of enzyme kinetics (Fig. 5-8-10) shows that SoUGT_2207 exhibits a distinct preference for SO1700 and UDP-GIc, resulting in the predominant formation of SO1861. Conversely, the addition of a glucose moiety onto SO1730, leading to SO1891 (Fig. 9-10), is observed as a minor product. Notably, SoUGT_10304 displays a clear preference for UDP-Xylose and SO1700, yielding SO1831. Kinetic analysis (Fig. 10) indicate that SoUGT_10304 exhibits a higher affinity for UDP-GIc, as evidenced by lower Km values compared to those for UDP-Xyl. Despite this, the overall production of SO1861 is significantly lower than that of SO1831. These findings underscore the existence of functional redundancy between different enzymes, such as SoUGT_2207 and SoUGT_10304, and substrate promiscuity. This leads to the generation of structurally related molecules in plant tissues, contributing to the increased complexity of the metabolic profile and posing challenges for conventional chromatographic purification strategies.

A schematic summary of enzymatic reactions by SoUGT_2207 and SoUGT_10304 is depicted in Figure 11.

Example 7: In-vitro production of SO1861 by the isolated S. officinalis UGTs using S.officinalis leaves extract metabolites

In the present example we tested the in-vitro production of SO1861 by evaluating the conversion of precursors from S.officinalis leaves extract to assess the possibility of using naturally sources as raw materials for SO1861 production. In vitro reactions were performed using 50 pg of purified SoUGT_2207 or SoUGT_10304 enzymes (described in Example 4) in the presence of either 100 pM UDP-GIc (uridine diphosphate glucose) or UDP-Xyl (uridine diphosphate xylose) as the sugar donor, and 10 uL of S.officinalis leaves extract as acceptor substrate. The latter was obtained as previously described (4). The final reaction volume was 100 pL, and the buffer contained 20 mM Tris-HCI and 15 mM MgCI₂ (pH 7.5), we calculated that the final concentration of the enzymes in the in vitro extract was 10 mM. The reactions were incubated at 30 °C overnight. The reactions were stopped by adding 100 pL of pure methanol and centrifuged at 13,500 rpm for 30 minutes to precipitate the enzymes. The supernatants were collected and submitted for HPLC analysis at the Core Mass Spectrometry Facility of the University of Edinburgh (EdinOmics), as in previous examples.

The results confirmed that SoUGT_10304 exhibits a degree of plasticity in sugar acceptor specificity, as it is capable of producing both SO1831 and SO1861 through the addition of a single molecule of UDP-Xyl or UDP-GIc, respectively (Fig. 13). In contrast, SoUGT_2207 demonstrated stricter specificity for the production of SO1861, utilizing UDP-GIc exclusively as the sugar donor. The incubation of the extract with SoUGT_2207 resulted in almost a 3-fold increase in SO1861 production compared to the basal levels observed in control reactions without enzyme addition.

Example 8: Heterologous expression of S. officinalis UDP-Glycosyltransferases enables SO1861 production in Gypsophila eleqans

In this example, various constructs were designed to achieve the ectopic overexpression of S.officinalis SoUGT 10304 and SoUGT 2207 in recombinant hairy roots of Gypsophila elegans, with the aim of evaluating their effects on the production levels of SO1861 and the related saponin SO1831. Gypsophila elegans was selected as the experimental system for the ease of genetic transformation compared to S. officinalis, thus enabling efficient generation of transgenic lines. Additionally, G. elegans plants have been reported to naturally produce compounds with high structural homology to SO1861, as well as comparable endosomal escaping properties (11).

Hence the expression of S. officinalis UGTs in G. elegans could activate and direct the biosynthetic pathway towards the production of SO1861 in this system, by leveraging newly discovered S. officinalis enzymes and the inherent biosynthetic capabilities of G. elegans. For this purpose, the coding sequences of both enzymes were inserted into the Gateway-compatible binary vector pK7WG2D (5).

The resulting transgenic G. elegans hairy root lines were generated via Agrobacterium rhizogenes- mediated transformation of G. elegans seedlings, following a previously established protocol (9). After selection, the transgenic hairy root cultures were grown for two months in liquid Murashige and Skoog medium containing vitamins and 1.5% sucrose. Subsequently, the hairy roots were harvested, flash- frozen in liquid nitrogen, and ground into a fine powder. The comprehensive extraction of metabolites and subsequent LC-MS analysis were carried out in accordance with previously documented procedures (6) employing total methanolic extraction from 100 mg of frozen powdered samples. The lyophilized methanolic extracts of hairy roots were sent to the Core Mass Spectrometry Facility of the University of Edinburgh (EdinOmics) for HPLC analysis. For each transgene three independent hairy root lines were cultured, analyzed and compared. Hairy roots of G. elegans hairy roots expressing S.officinalis beta- amyrin synthase (4), an enzyme catalyzing the production of the early pathway precursor beta-amyrin from epoxy-squalene, were used as control lines.

The analysis revealed that transgenic lines for all three transgenes exhibited comparable levels of SO1831. By either "pushing" the metabolic flux through the saponin pathway via increased precursor abundance (achieved through SoBAS overexpression) or "pulling" it by expressing new-late pathway enzymes, SoUGT_10304 or SoUGT_2207, it was possible to achieve consistent SO1831 production (Fig. 14). In contrast, the specific activity of SoUGT_2207 towards UDP-GIc and triterpenoid substrates resulted in a significantly higher production of SO1861, with an almost three-fold increase compared to the other two lines (Fig. 14).

Sequence listing

SEQ. ID NO: 1 - S. officinalis Glycosyltransferase SoUGT_2207 coding sequence (1497 bps)

ATGGACTCGAATTCTAACAACGACGACGACAACAGAGTGAAGATGTTTTTCTTCCCATACATAGCAGGTGGTCAT CTTCTACCAATGATTGACCTTGCCAAATTTTTTGCATCGTCTCACCCTCTCGTTGACGCCACCGTCATCACCACCCC TAAAACCGCCGCCCTCTTCCAATCCTCCTCCTCCTCACCCATCCCCAACTTCTCCTTCCTCACCCTAGAGCTCCCTAA AAACGACAACGACAACGACGAGGATAACGACGAGAACTTCCTTAGTACAATGTCTGCTGACCAAAGAGCTAAAG

TCTTAGCATCCTTTAAAAATCTCAAAGAACCCCTTCAGAAATTACTCTTCGATTTAAAACCGCATTGTTTCGTTAGC

GATTTGCTGCATTCGTGGACGATTGAACTTGCTCAAAACGCCGGTGTTCCGTGGGTTGTTTTTCATAGTACTTGTT

TGTTTGTGTTGTGTGTCGAGGACTACTTGGCTCGGTTTAAGCCTCACCGTAAAGGGGGTTTGGATTCTGATATGT

TTTTACTAAGCGGGTTGAAAGACGACTCGGTTAGATTCAATAAATTACGCTTACCGGTTTGGCATCGTGGCGATG

AAATTCCGGAAGCGTTTCTACCCATTAATTTGATTCGAAAGGCGTGTTCTAGGAGTTACGCTATGGTCGTGAATA

GTTCTTTCGAGTTTGAAGGAGAGTTCCGTGAACGTGTTCAGAACGCGGTTGGGGCTCCTCGTGTTTCGATGGTG

GGCCCAGTGAGCTTAAACTCGGCCAGGAACGTGAGCGATGCGAAGGTCGAGAATGTGAAGAGCGATATCATAC

TCAAATGGCTTAATTTGAATCAACCGGGTTCGGTCGTGTACGTGAGTTTCGGGAGCGAGGCGAATTTGTGTAAG

GGCCAATTTCACGAAATCGCTCATGGGTTAGAGAGTTCGGGTCAACCCTTTATTTGGGTGGTTAGGCCCAATTTG

TTTAAAGCCCAAGGCGACGAGGGGTGGTTCCCTGAAGGGTTCGAGTCCCGAGTAAGGGACTCGAACCAAGGGC

TAATAATCAAAGAGTGGGCCCCCCAACTTGTTATATTAGGTCATGTTAGTCTAGGCGCGTTTGTGACTCACTGTG

GCTGGAACTCGGTTCTGGAAGGGTTAAGCAATGGGGTGCCCATGATCACATGGCCACTAACCCATGACCAATTTT

ACGTCGAGAGCTTGATTGTGGATGTGTTGAAGGTCGGGCTTAGGGTCGGGAATGAGGAGTGGGTCGATATCAT

TTGGCCACCAAAGGTGGCCGTGACACGAGATCAGGTCGAGACAGCAGTGAGACAGATGATGGGTGGGCGGGA

CGAGGTGGAAGAGATGAGGAGAAATGTGAAGGAATATGCTAACAAGTGTAAGATGAGTGTTCAAGAAGGAGG

ATCTTCATATGAAGATGTGTGTGCACTTGTTGAGGAACTCAAAGCTCATAGAAATGAATTGTCACAAAAACTAGT CTAA

SEQ ID NO: 2 - S. officinalis Glycosyltransferase SoUGT_2207 translated nucleotide sequence (498 AA)

MDSNSNNDDDNRVKMFFFPYIAGGHLLPMIDLAKFFASSHPLVDATVITTPKTAALFQSSSSSPIPNFSFLTLELPKND

NDNDEDNDENFLSTMSADQRAKVLASFKNLKEPLQKLLFDLKPHCFVSDLLHSWTIELAQNAGVPWVVFHSTCLFVL

CVEDYLARFKPHRKGGLDSDM FLLSGLKDDSVRFNKLRLPVWHRGDEIPEAFLPINLIRKACSRSYAMVVNSSFEFEGE

FRERVQNAVGAPRVSMVGPVSLNSARNVSDAKVENVKSDIILKWLNLNQPGSVVYVSFGSEANLCKGQFHEIAHGLE

SSGQPFIWVVRPNLFKAQGDEGWFPEGFESRVRDSNQGLIIKEWAPQLVILGHVSLGAFVTHCGWNSVLEGLSNGV

PMITWPLTHDQFYVESLIVDVLKVGLRVGNEEWVDIIWPPKVAVTRDQVETAVRQMMGGRDEVEEM RRNVKEYA

NKCKMSVQEGGSSYEDVCALVEELKAHRNELSQKLV

SEQ ID NO: 3 - S. officinalis Glycosyltransferase SoUGT_10304 coding sequence (1473 bps)

ATGGAGGAATCAAAGGAGGAAGTACATGTAGCATTCTTCCCATTCATGACACCAGGTCACTCAATCCCAATGCTA

GACTTGGTACGTTTGTTCATTGCTCGTGGTGTCAAAACTACTGTCTTCACTACTCCTCTTAATGCTCCTAATATTTC

CAAATACCTCAACATTATCCAAGATTCCTCATCAAACAAAAACACCATTTATGTAACTCCTTTTCCTTCTAAAGAAG

CCGGTTTACCGGAAGGTGTGGAAAGCCAGGATAGTACCACTTCCCCCGAAATGACCCTCAAGTTCTTTGTTGCTA

TGGAATTACTTCAAGACCCCCTTGATGTTTTTTTAAAAGAAACCAAACCTCATTGTCTTGTTGCTGATAATTTCTTC CCTTACGCCACCGACATCGCTTCTAAGTATGGCATTCCTAGGTTTGTTTTTCAGTTCACTGGCTTCTTTCCTATGTCT GTCATGATGGCCTTAAATCGTTTCCACCCTCAAAACTCTGTATCATCTGATGACGACCCCTTTCTTGTTCCCAGTTT

ACCCCATGACATCAAATTGACTAAGTCACAATTGCAACGAGAGTACGAGGGTAGTGATGGTATTGACACCGCTCT

TTCTAGGCTCTGTAATGGCGCCGGTAGAGCTTTGTTTACTAGTTATGGTGTCATTTTTAACAGCTTCTACCAACTC

GAACCTGATTATGTTGATTATTATACCAACACCATGGGGAAACGATCCAGGGTTTGGCATGTGGGCCCAGTGTCG

TTATGCAACCGTCGACACGTGGAGGGTAAATCTGGTAGGGGGAGAAGTGCTTCAATTAGTGAGCATTTGTGCTT

AGAGTGGCTCAATGCCAAAGAACCAAATTCAGTGATATATGTATGTTTTGGTAGTCTCACATGTTTCTCCAATGA

GCAACTCAAAGAAATCGCAACCGCCTTAGAAAGGTGTGAAGAGTATTTTATATGGGTGTTGAAGGGTGGCAAAG

ATAATGAGCAAGAGTGGTTGCCACAAGGGTTTGAAGAGAGGGTTGAAGGGAAAGGACTAATCATACGGGGGT

GGGCCCCACAAGTGTTGATTTTAGACCATGAAGCCATAGGCGGGTTTGTGACACACTGTGGTTGGAACTCGACA

CTAGAAAGTATATCAGCGGGGGTGCCCATGGTGACATGGCCCATATATGCAGAGCAATTTTATAATGAGAAATT

GGTGACGGATGTACTGAAGGTGGGGGTTAAAGTAGGGTCAATGAAGTGGAGTGAGACGACGGGGGCGACTCA

TTTAAAGCATGAGGAAATAGAAAAAGCATTGAAGCAAATAATGGTGGGAGAAGAGGTGTTAGAGATGAGAAAA

AGAGCAAGTAAGTTGAAAGAGATGGCTTATAATGCTGTTGAAGAAGGAGGCTCTTCTTATTCTCACCTCACTTCC

TTAATCGACGACCTTATGGCTTCCAAAGCTGTGCTACAAAAATTTTGA

SEQ. ID NO: 4 - S. officinalis SoUGT_10304 translated nucleotide sequence (490 AA)

MEESKEEVHVAFFPFMTPGHSIPM LDLVRLFIARGVKTTVFTTPLNAPNISKYLNIIQDSSSNKNTIYVTPFPSKEAGLPE

GVESQDSTTSPEMTLKFFVAM ELLQDPLDVFLKETKPHCLVADNFFPYATDIASKYGIPRFVFQFTGFFPMSVM MALN

RFHPQNSVSSDDDPFLVPSLPHDIKLTKSQLQREYEGSDGIDTALSRLCNGAGRALFTSYGVIFNSFYQLEPDYVDYYTN

TMGKRSRVWHVGPVSLCNRRHVEGKSGRGRSASISEHLCLEWLNAKEPNSVIYVCFGSLTCFSNEQLKEIATALERCEE

YFIWVLKGGKDNEQEWLPQGFEERVEGKGLIIRGWAPQVLILDHEAIGGFVTHCGWNSTLESISAGVPMVTWPIYAE

QFYNEKLVTDVLKVGVKVGSMKWSETTGATHLKHEEIEKALKQIMVGEEVLEMRKRASKLKEMAYNAVEEGGSSYSH

LTSLIDDLMASKAVLQKF

References

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2. Buntru M, Hahnengress N, Croon A, Sch ill berg S. 2022. Plant-Derived Cell-Free Biofactories for the Production of Secondary Metabolites. Front Plant Sci 12:794999

3. Buntru M, Vogel S, Stoff K, Spiegel H, Schillberg S. 2015. A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates. Biotechnol Bioeng 112:867- 78

4. Jo S, El-Demerdash A, Owen C, Srivastava V, Wu D, et al. 2024. Unlocking saponin biosynthesis in soapwort. Nat Chem Biol

5. Karimi M, Inze D, Depicker A. 2002. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193-5

6. Mertens J, Pollier J, Vanden Bossche R, Lopez-Vidriero I, Franco-Zorrilla JM, Goossens A. 2016. The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula. Plant Physiol 170:194-210

7. One Thousand Plant Transcriptomes I. 2019. One thousand plant transcriptomes and the phylogenomics of green plants. Nature 574:679-85

8. Patro R, Duggal G, Love Ml, Irizarry RA, Kingsford C. 2017. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417-9

9. Pollier J, De Geyter N, Moses T, Boachon B, Franco-Zorrilla J M, et al. 2019. The MYB transcription factor Emission of Methyl Anthranilate 1 stimulates emission of methyl anthranilate from Medicago truncatula hairy roots. Plant J 99:637-54

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Claims

Claims

1. A plant extract comprising: a. between 1 mM to 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, b. and/or including 1 mM and 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4, c. and 10 mM to 500 mM of UDP-glucose.

2. A plant extract according to claim 1 further comprising sapofectosid precursors SQ1700 and/or SO1730.

3. A plant extract according to claim 1 or 2 wherein said plant is selected from the family of Caryophyllaceae plants.

4. A plant extract according to claim 3 wherein said plant is selected from the genus Saponaria, Gypsophila or Agrostemma.

5. A plant extract according to claim 1 or 2 wherein said plant is from the genus Quillaja.

6. A method to produce sapofectosid comprising adding to an in vitro system between 1 mM to 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, optionally also including 1 mM and 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: A, 10 mM to 500 mM of UDP-glucose and sapofectosid precursors SQ1700 and/or SQ1730.

7. A method according to produce sapofectosid comprising adding to a plant extract between 1 mM to 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, and/or including 1 mM and 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4 and 10 mM to 500 mM of UDP-glucose.

8. A method according to claim 7 further adding sapofectosid precursors SQ1700 and/or SQ1730.

9. A method according to claims 7 or 8 wherein said plant is selected from the genus Saponaria, Gypsophila, Agrostemma or Quillaja.

10. A nucleotide sequence encoding a protein having at least 70% amino acid identity over the total length of SEQ ID NO: 2.

11. A protein sequence having at least 70% amino acid identity over the total length of SEQ ID NO: 2.

12. A Saponaria or Gypsophila plant, plant cell or hairy root having a gene disruption in the gene encoding for an amino acid having at least 70% amino acid identity over the total length of SEQ ID NO: 4 and said plant, plant cell or hairy root comprising a chimeric gene encoding for a protein having at least 70% amino acid identity over the total length of SEQ ID NO: 2.

13. An in vitro system comprising: a. between 1 mM to 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 2, b. and/or including 1 mM and 100 mM of an enzyme having at least 70% amino acid identity over the total length of SEQ ID NO: 4, c. 10 mM to 500 mM of UDP-glucose, and d. sapofectosid precursors SQ1700 and/or SQ1730.