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WO2024047057A1 - Moyens et procédés de production de saponines triterpéniques dans des cellules eucaryotes - Google Patents

Moyens et procédés de production de saponines triterpéniques dans des cellules eucaryotes Download PDF

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WO2024047057A1
WO2024047057A1 PCT/EP2023/073700 EP2023073700W WO2024047057A1 WO 2024047057 A1 WO2024047057 A1 WO 2024047057A1 EP 2023073700 W EP2023073700 W EP 2023073700W WO 2024047057 A1 WO2024047057 A1 WO 2024047057A1
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seq
sop450
yeast
plant
nucleotide sequence
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Alain Goossens
Elia LACCHINI
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Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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Vlaams Instituut voor Biotechnologie VIB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99039—Beta-amyrin synthase (5.4.99.39)

Definitions

  • 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.
  • 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.
  • 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).
  • Glc D-glucose
  • Gal D-galactose
  • GlcA D-glucuronic acid
  • GalA D-galacturonic acid
  • L-rhamnose Rha
  • L-arabinose Ara
  • D-xylose Xyl
  • 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.
  • 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.
  • 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 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 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 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.
  • biosynthetic enzymes in Saponaria officinalis which enzymes when expressed as chimeric genes in an eukaryotic host, lead to the production of quillaic acid.
  • quillaic acid can be produced in the yeast Saccharomyces cerevisiae by expression of the identified chimeric genes.
  • the isolated biosynthetic enzymes comprise an unusual combination of a dual specificity C-16/C-28 oxidase and two different C-23 oxidases.
  • 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 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.
  • 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.
  • the eukaryotic cell further comprises a chimeric gene encoding a NADPH- cytochrome P450 reductase.
  • a NADPH-cytochrome P450 oxidase is depicted in the nucleotide sequence SEQ ID NO: 15.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the yeast cell further comprises a chimeric gene encoding an amino-terminal truncated feedback-insensitive 3-hydroxy-3-methylglutaryl coenzyme A reductase.
  • 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.
  • 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.
  • said plant cell is from the genus Saponaria or Gypsophila.
  • 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).
  • the promoter is an inducible promoter.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the method to produce quillaic acid in yeast further comprises the introduction of a chimeric gene encoding a NADPH-cytochrome P450 reductase.
  • 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.
  • operably linked 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.
  • terminal 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.
  • 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.
  • 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).
  • antibiotics such as nptll that phosphorylates
  • 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).
  • colour for example p-glucuronidase, GUS or p- galactosidase with its coloured substrates, for example X-Gal
  • luminescence such as the luciferin/luciferase system
  • fluorescence Green Fluorescent Protein
  • 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).
  • 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.
  • the transformants usually receive only a part of the vector, i.e.
  • the marker genes can subsequently be removed from the transformed plant by performing crosses.
  • 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.
  • the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost.
  • the transposon jumps to a different location. In these cases the marker gene must be eliminated by performing crosses.
  • Cre/lox 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.
  • 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.
  • transgenic 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.
  • 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.
  • 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.
  • 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.
  • sequence alignment 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.
  • expression means the transcription of a specific gene or specific genes or specific genetic construct.
  • 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.
  • 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 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.
  • 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.
  • Transformation of plant species is now a fairly routine technique.
  • 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.
  • Transgenic plants including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation.
  • An advantageous transformation method is the transformation in planta.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 (PromegaTM), 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 (QuantaBioTM) and for the cloning of candidate genes. The S.
  • transcripts levels were estimated by mapping single-end reads on the S. officinalis transcriptome using Salmon [2] implemented on a Galaxy pipeline.
  • 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.
  • SOM Self Organizing Map
  • SoBAS_1421 a candidate p-amyrin synthase 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.
  • SoBAS_1421 full coding sequence and predicted protein are reported as SEQ ID NOs: 1 and 2.
  • P450s cytochrome P450s
  • SoBAS_1421 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.
  • SoP450_1944 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.
  • SoP450_1049 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.
  • SoP450_2010 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.
  • SoP450_6085 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.
  • SEQ ID NOs: 9 and 10 The full nucleotide and predicted protein sequences of SoP450_6085 are reported as SEQ ID NOs: 9 and 10.
  • UDP-glycosyltransferase (UGT) encoding gene SoUGT_2488 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 (2016) 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).
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • Strain 4 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).
  • SoP450_1944 corresponds to a p-amyrin C-28 oxidase.
  • strain 5 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.
  • 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).
  • gypsogenin is characterized by aldehyde function at position 23 while the same carbon is further oxidized to carboxy group in gypsogenic acid.
  • SoP450_2010 is able to accept oleanolic acid as a substrate and confirm its activity to be specifically oxidizing the carbon at position 23.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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
  • 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).
  • Example 5 Overexpression of newly discovered enzymes in transgenic Gypsophila elegans hairy root lines
  • 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.
  • 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).
  • the Gateway-compatible binary vector pK7WG2D was utilized, as outlined in Example 4 for S. officinalis hairy roots .
  • three separate hairy root lines were cultivated, analyzed, and compared against three distinct control lines (expressing solely the metabolically inactive green fluorescent protein, GFP).
  • SEQ ID 21 SoSQE2 Saponaria officinalis, Squalene Epoxidase 2 coding sequence (1557 bps)
  • Goossens A Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16alpha hydroxylase from Bupleurum falcatum. Proc Natl Acad Sci U S A 2014, 111:1634-1639.

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Abstract

La présente invention concerne de nouveaux gènes chimériques utiles à la production d'acide quillaïque et de dérivés associés dans des cellules eucaryotes telles que celles des levures et des plantes.
PCT/EP2023/073700 2022-09-01 2023-08-29 Moyens et procédés de production de saponines triterpéniques dans des cellules eucaryotes Ceased WO2024047057A1 (fr)

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EP4453228A4 (fr) * 2021-12-24 2025-09-10 Univ California Production de saponine dans levure

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