Production and secretion of secondary metabolites by hairy roots

Abstract

The method comprises a step of culture of genetically engineered hairy roots in a culture medium, a step of elicitation of the production of a secondary metabolite of interest; optionally a step of induction of the secretion of the secondary metabolite into the culture medium by addition of an agent, and a step of collection of the culture medium containing the secreted metabolite of interest with addition of fresh culture medium, wherein the steps of secretion and collection are repeated sequentially.

Description

PRODUCTION AND SECRETION OF SECONDARY METABOLITESBY HAIRY ROOTSFIELD OF INVENTION

[0001] The present invention relates to a method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system.BACKGROUND OF INVENTION

[0002] Hairy roots from plants have been widely studied and used for the production of secondary metabolites of industrial and pharmaceutical interest. The production of secondary metabolites can be performed from hairy roots of selected plants obtained after infection with Rhizobium rhizogenes, a naturally occurring soil bacterium. During the infection of the plant, R. rhizogenes transfer part of its DNA, called T-DNA which includes the plasmid Ri, to the plant, thus causing the emergence of hairy roots at the infection site. The hairy roots can later be isolated from the plant and cultured independently either on solid or in liquid culture medium.

[0003] However, the natural ability of wild-type hairy roots to produce secondary metabolites in standard culture conditions is limited. Genetic engineering was therefore introduced as a first effort to improve the production of secondary metabolites in hairy roots. More precisely, R. rhizogenes is genetically modified so that after infection, the synthesis pathway of secondary metabolites in the plant is altered, and their production increased.

[0004] To improve the production of secondary metabolites, it is also possible to modify the hairy root culture conditions in order to mimic a stress, to which the plants would be subjected in their natural environment. Such stress will elicit a response in the hairy roots, notably the synthesis of secondary metabolites. This method called “elicitation” has beenshown to yield good results in improving the production of secondary metabolites in hairy roots.

[0005] However, even with all these possible improvements, the rate of production of secondary metabolites remains limited. Each method used to boost the production has an impact on the condition of the hairy root and may reduce its longevity. Thus, there is a need to improve the yield of secondary metabolites production, while preserving the hairy roots, so that several cycles of collection can be undergone consecutively, thus saving time, energy and costs.

[0006] The method of the invention improves the rate of production of compounds while allowing a continuous production. The method of the invention combines genetic engineering, and elicitation, to induce the secretion of the compound out of the root, in large quantities. The final step of collection may be repeated easily via a simple renewal of culture medium and without having to start a new growth phase (Figure 1).

[0007] Thus, the method of the invention allows the person skilled in the art to re-use the hairy roots, and leads to a similar or even higher metabolite production per g of biomass ratio. Such an improvement is very advantageous in term of energy costs and saves time. It is therefore more sustainable than previously used methods.SUMMARY

[0008] The invention relates to a method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system, the method comprising: a) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction;b) a step of elicitation of the production of a secondary metabolite of interest by applying an elicitor; c) optionally a step of induction of the secretion of the secondary metabolite into the culture medium by addition of an agent; and d) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium, wherein at least said steps c) and d) are repeated sequentially at least one time.

[0009] In some embodiments, the steps b), c) and d) are repeated sequentially at least one time.

[0010] In some embodiments, the method does not comprise any step in which the hairy roots are incubated in a solution comprising from about 250 mM to about 4M of a salt.

[0011] In some embodiments, the secreted secondary metabolite is selected in the group consisting of: polyphenols, alkaloids, cannabinoids, terpenoids, steroids, saponins, flavonoids, and tannins.

[0012] In some embodiments, the genetically engineered hairy roots are obtained by introducing a nucleic acid encoding for at least one gene involved in a plant metabolic pathway.

[0013] In some embodiments the elicitor is selected from the group consisting of : plant hormones, such as, e.g., auxins, and the like; phytopharmaceuticals, such as, e.g., methoxyfenozide, tubefenozide and the like; steroids, such as, e.g., dexamethasone, estradiol, and the like; alcohols, such as, e.g., ethanol, methanol, and the like; metal ions, such as, e.g., copper, silver, cadmium, cobalt, and the like; antibiotics such as, e.g., tetracycline; polyosides, such as, e.g., cyclodextrins, chitosan, chitin, sucrose, sorbitol, dextran, and the like; polypeptides, such as, e.g., elicitin; inorganic salts, such as, e.g., sodium orthovanadate, vanadyl sulphate, sodium chloride, and the like; organic salts, such as, e.g., methyl jasmonate, jasmonic acid, gibberellic acid, salicylic acid, sodiumsalicylate, abscisic acid and the like; proline; organic molecule, such as, e.g., 2,4- dichlorophenoxyacetic acid, polyethylene glycol, tween, PVP (polyvinylpyrrolidone), urea, and the like; yeast extracts; microorganisms, such as, e.g., Trichoderma atroviride, Protomyces gravid s, Claviceps purpurea, Mucor hivemalis, Fusarium oxysporum, Phoma exigua, Botrytis cinerea, Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli, Rhizobium leguminosarum; red light, green light, blue light, temperature, oxygen, pH, ozone, UV-C, osmotic stress, and combinations thereof.

[0014] In some embodiments, the elicitor is selected from the group consisting of: methyl jasmonate, jasmonic acid, chitosan, salicylic acid, jasmonate, cadmium chloride (CdC12), coumarine or furocoumarine, cyclodextrin, gibberellic acid, yeast extract, Phytopthora parasitica filtrate, Aspergillus niger, cellulase, 2,4-dichlorophenoxyacetic acid and Bacteria sp.

[0015] In some embodiments, the elicitor is an abiotic elicitor selected from the group consisting of red light, green light, blue light, temperature, oxygen, pH, ozone, UV-C, osmotic stress, and combinations thereof.

[0016] In some embodiments, the elicitor is an exposition of the hairy roots to UV, preferably UV-A and / or UV-B.

[0017] In some embodiments, the hairy roots are incubated in the culture medium with the elicitor at step b) for about 3 days to about 30 days, preferably for about 3 days to about 20 days.

[0018] In some embodiments, the secretion is induced by ethanol, chitosan, salicylic acid, jasmonic acid, Tween20, pH (2<pH< 8).

[0019] In some embodiments, the secretion lasts for about 1 day to about 60 days, preferably for about 1 day to about 20 days.

[0020] In some embodiments, the hairy roots belong to the species Brassica rapa rapa, Brassica napus, Salvia Milthiorrhiza, Panax Ginseng, Armoracia rusticana, Trigonella foenumgraceum, Lippia dulcis, Lithospermum erythrorhizon, Ophiorrhiza pumila, and. Echinacea purpurea, Echinacea Angustifolia, Puerariaphaseoloides, Harpagophytum Procumbens, Morinda Citrifolia, Hypericum Perforatum, Derris trifolia, Salvia miltiorrhiza, Salvia prevalzkii, Echinacea pallida, Cistanche tubulosa, Glycyrrhiza glabra, Sophora flavescens, Rhodiola Rosea, Polygonum cuspidatum, Fallopia multiflora, Lepidium peruvianum, Whitania Somnifera, Astragalus Membranaceous, Berberis Vulgaris, Sanguinaria canadensis, Eleutherococcus Senticosus, Cannabis sativa, Hydrastis Canadensis, Arctium Majus, Piper methysticium, Pueraria lobata, Glycyrrhiza uralensis, Ptychopetalum olacoides, Dioscorea Vollosa, Yucca shidigera, Panax quinquefolius, Azadirachta indica, Catharanthus trichophyllus, Calystegia sepium, Atropa belladonna, Hyoscyamus muticus, Duboisia myoporoides, Duboisia leichhardtii, Artemisia annua, Datura stramonium, Arabidopsis thaliana, Stizolobium, Hassjoo, Ipomea aquatica, Perilla fruitescnens, Catharanthus roseus, Taxus brevifolia, Gloriosa Superba, Saponaria officinalis, Solanum tuberosum, Nicotiana tabacum, Nicotiana benthamiana or Cinchosa Pubescens, preferably to the Brassica rapa rapa or Brassica napus species.DEFINITIONS

[0021] In the present invention, the following terms have the following meanings:

[0022] “Continuous production” refers to a hairy roots-based expression system allowing the production of secondary metabolites without interruption. A continuous production allows to achieve several cycles of culture and collection of a metabolite of interest without any interruption. Continuous production may be performed according to the invention because the root biomass is not destroyed after the steps leading to the collection of the metabolites. In other words, a continuous production refers to a method that is ongoing and does not involve intermittent or batch processing. It is understood that during the repeated steps the hairy roots-based expression system continues to produce the secondary metabolites of interest. Illustratively, upon the step of collection (i) of the secondary metabolites of interest, the hairy roots may be washed with water or culturemedium or the culture medium could be just harvested and, be submitted to another step of induction of the secretion (step c) so as to start another cycle of production and collection. In practice, a fresh culture medium is added to the hairy root biomass and a new round of production of the secondary metabolites of interest can be initiated.

[0023] “Secondary metabolites” alternatively defined as “specialized metabolites” refer to small molecules i.e., intermediate or final products of metabolic reactions, such as, e.g., polyphenols, alkaloids, cannabinoids, terpenoids, saponins, steroids, flavonoids and tannins. Valuable metabolites, i.e., metabolites of interest, may be naturally synthesized and secreted by plants, in particular by roots and hairy roots. Alternatively, the synthesis and secretion of valuable metabolites may be artificially induced in hairy roots. According to the present invention, the secondary metabolite is produced by the genetically engineered hairy root-based system, especially obtained following a genetical modification of the synthesis pathway of said secondary metabolite.

[0024] “Secreted metabolite” refers to a metabolite which, upon synthesis in the cells of the root system, in particular of the hairy root system, crosses the cellular membrane / envelop and is to be localized outside these cells.

[0025] “Hairy roots-based expression system” (also sometimes referred to as “hairy root system” or “hairy root expression system”) refers to a culture of hairy roots, either previously engineered (for example genetically modified) or not, so that they can synthesize a compound of interest, allowing in fine the hairy roots to produce said compound.

[0026] “Hairy root” refers to root emergences which appear after the infection of a plant by Rhizobium rhizogenes (previously referred to as Agrobacterium rhiz.o genes) bacteria or by Rhizobium radiobacter (also known as Agrobacterium Tumefaciens) bacteria harboring rol genes for example.

[0027] “Adventitious roots” refers to root emergences which appear during the physiological development of the plant and hence occurs naturally.

[0028] “Culture medium’’ refers to a liquid medium containing all the required nutrients in which the hairy roots, are cultivated. As used herein, the term “culture medium” is a substance containing nutrients in which hairy roots can be maintained and / or grown. Culture media thus contain all the elements that the hairy roots, need for survival and / or growth. An undefined medium may comprise a carbon source, water, salts, a source of amino acids and a source of nitrogen.

[0029] “Genetically engineering” refers to the genetic modifications carried out on an organism so as to improve the production of a metabolite of interest, especially here to modify and / or reinforce the synthesis pathway of secondary metabolites in the plant, to improve their production. An example of modification is the overexpression of transcription factors or enzymes of the synthesis pathway or inhibition of competitive pathways.

[0030] Biomass growth” may refer to the process of culturing a plant, in particular hairy roots, in order to increase its weight, as measured for example by its weight of dry or fresh matter. “Biomass growth” may also refer to the production or increase of biomass, for example of hairy root biomass, over the course of a culture, for example of a hairy root culture.

[0031] “Elicitation” refers to the addition of molecule(s) called “elicitors”, or the application of specific physical parameter (temperature, light, UV, osmotic pressure etc.) which will act as a stimulator of the plant’s natural defenses, including the production of secondary metabolites. Different types of elicitors can be used in hairy root culture to improve the synthesis of secondary metabolites, such as biotic elicitor (for example yeast extract, fungal extract or living bacteria) or abiotic elicitors (such as osmotic stress, jasmonic acid methyl jasmonate, salicylic acid or chitosan for signal molecules). The elicitation increases the amount of secondary metabolite inside the roots and in some cases, in the culture medium. One example of elicitor is 2,4-dichlorophenoxyacetic acid.

[0032] “Osmotic pressure” or “osmotic stress” refers here to the application of a salt in small concentration to the culture medium (and always below 250mM), in order to induce a stress to the root. In the context of the invention, an osmotic pressure is an elicitor and the aim of provoking such stress is to stimulate the production of secondary metabolite by the plant. As for the other types of elicitors, the incubation of the roots with salt causing the osmotic pressure lasts a minimum of 3 days and up to 60 days.

[0033] “Recovery” is intended to mean that the secondary metabolites of interest, which is obtained by the method according to the invention, in particular the hairy root system, is physically separated from the root system itself. In some embodiments, the terms “recovery”, “elution”, “harvest”, “collection” or “obtention” may be substituted to one another.

[0034] “Step of culture” or “Growth phase” or “Phase of culture” refers to the phase of culture of the roots, in particular the hairy roots, wherein the roots, in particular the hairy roots, are cultured within an appropriate culture medium in order to maintain the roots, in particular the hairy roots, in a state in which the cells of the roots, in particular of the hairy roots, may divide, have an active metabolism, increase their overall biomass and / or produce a compound of interest.

[0035] “Rhizocals induction” refers to a phase of culture of the roots, in particular of the hairy roots, wherein the culture medium is supplemented with an agent, for example a hormone, more specifically an auxin, such as, e.g., 2.4-D, which is capable of promoting a biomass growth cessation in the same time as an induction of rhizocals. The rhizocals, which correspond to a modification of the structure of the roots, enable an increase of the ability of the roots to produce and / or secrete a compound of interest.

[0036] “Secretion” refers to the process by which a secondary metabolite produced within a hairy roots (or rhizocal) cell crosses the cellular envelop and is to be localized outside the cell. In the context of the invention, the secretion is induced by addition of anagent such as ethanol, chitosan, salicylic acid, methyl jasmonate, jasmonic acid, yeast extract, Tween20, pH.

[0037] “About” preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers to is itself also specifically, and preferably, disclosed.

[0038] “Comprise” is intended to mean “contain”, “encompass” and “include”. In some embodiments, the term “comprise” also encompasses the term “consist of’.

[0039] “Rhizocal” or “rhizocallus” refers to a conic-shaped structure connected to the roots, in particular to the hairy roots, also termed lateral root emergence, which develops alongside of the roots in a solidarized way. In practice, rhizocals (or “rhizocalli”) may be induced in a culture of roots, in particular in a culture of hairy roots, by the addition in the culture medium of one or more agent(s) that promote(s) the induction of rhizocals such as, for example, the hormone called auxin or synthetic auxins such as 2,4- dichlorophenoxyacetic acid (2,4-D). The presence of “rhizocals” in a culture of hairy roots is often associated with a better yield in the production of a compound of interest by the hairy roots.

[0040] The term “yield”, as known in the art, refers to the amount of the secondary metabolite of interest recovered upon the method according to the invention.DETAILED DESCRIPTION

[0041] This invention relates to a method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system, the method comprising: a) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction;b) a step of elicitation of the production of a secondary metabolite by applying an elicitor; c) optionally a step of induction of the secretion of the secondary metabolite by addition of an agent; and d) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium, wherein at least said steps c) and d) are repeated sequentially at least one time.

[0042] This invention also relates to a method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system, the method comprising: a) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction; b) a step of elicitation of the production of a secondary metabolite by applying an elicitor; and d) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium, wherein at least said steps b) and d) are repeated sequentially at least one time.

[0043] In some embodiments, steps b) and d) are carried out simultaneously. In some embodiments, steps b) and d) are carried out separately.

[0044] In some embodiments, the step (a) of culture of genetically engineered hairy roots in a culture medium allows the culture of the hairy roots dedicated to biomass growth, thereby allowing the production of the secondary metabolite of interest in a culture medium by said hairy roots, optionally in a culture medium suitable for rhizocal induction.

[0045] In some embodiments, the step (a) of culture of the genetically engineered hairy roots in a culture medium is dedicated to biomass growth.

[0046] In some embodiments, the hairy roots biomass growth is initiated during step (a) and continues during the following steps.

[0047] In some embodiments, during the step (a) of culture of the genetically engineered hairy roots, the secondary metabolite of interest is produced by the hairy roots.

[0048] Hairy root-based expression systems, have been abundantly described in the state of the art. In some embodiments, the root-based expression system, in particular the hairy root-based expression system used in the context of the invention may be known from the state of the art or be a system adapted or derived therefrom.

[0049] In certain embodiments, the roots are selected from a group consisting of adventitious roots, hairy roots, rhizocals, and any combination thereof, preferably, the roots are hairy roots.

[0050] In some embodiments the hairy roots or rhizocals belong to the species Brassica rapa rapa, Brassica napus, Salvia milthiorrhiza, Panax Ginseng, Armoracia rusticana, Trigonella foenumgraceum, Lippia dulcis, Lithospermum erythrorhizon, Ophiorrhiza pumila, and Echinacea purpurea, Echinacea angustifolia, Puerariaphaseoloid.es, Harpagophytum Procumbens, Morinda Citrifolia, Hypericum Perforatum, Derris trifolia, Salvia miltiorrhiza, Salvia prevalzkii, Echinacea pallida, Cistanche tubulosa, Glycyrrhiza glabra, Sophora flavescens, Rhodiola Rosea, Polygonum cuspidatum, Fallopia multiflora, Lepidium peruvianum, Whitania somnifera, Astragalus membranaceous, Berberis vulgaris, Sanguinaria canadensis, Eleutherococcus senticosus, Cannabis sativa, Hydrastis canadensis, Arctium majus, Piper methysticium, Pueraria lobata, Glycyrrhiza uralensis, Ptychopetalum olacoides, Dioscorea vollosa, Yucca shidigera, Panax quinquefolius, Azadirachta indica, Catharanthus trichophyllus, Calystegia sepium, Atropa belladonna, Hyoscyamus muticus, Duboisia myoporoides, Duboisia leichhardtii, Artemisia annua, Datura stramonium, Arabidopsis thaliana, Stizolobium, Hassjoo, Ipomea aquatica, Perillafruitescnens, Catharanthus roseus, Taxusbrevifolia, Gloriosa superba, Saponaria officinalis, Solanum tuberosum, Nicotiana tabacum, Nicotiana benthamiana or Cinchosa Pubescens, preferably to the Brassica rapa rapa or Brassica napus species.

[0051] In some embodiments, the hairy roots or rhizocals belong to a hybrid of two or more of the previously listed plant species.

[0052] In some embodiments, the hairy roots-based expression system is obtained by the infection of a plant by a suitable bacterial or viral strain, preferably a bacterial strain of Rhizobium rhizogenes (formerly known as Agrobacterium rhizo genes) or strain of Agrobacterium Tumefaciens harboring rol genes. This bacterial strain may comprise a vector containing an expression cassette comprising a gene encoding a protein involved in the metabolic pathway of the secondary metabolite of interest.

[0053] Within the scope of the invention, the term “expression cassette” refers to a nucleic acid construct which can be introduced in a cell and which allows the expression of the gene comprised in the expression cassette. In practice, a suitable expression cassette may comprise a promoter, a nucleic acid encoding a gene that will modify a plant’s metabolic pathway and consequently increase the secondary metabolite of interest’s expression, a terminator, optionally a signal peptide and, optionally regulatory sequences that allow controlling the steps of transcription (e.g., polyA sequence) and / or translation. Several expression cassettes can be integrated in one “molecular construct” before being introduced in a same hairy root.

[0054] In some embodiments, during step a), a molecular construct is introduced into bacteria before said bacteria are used to induce the formation of hairy roots in a plant, wherein said nucleic acid construct comprises at least one expression cassette, said expression cassette comprising: a promoter; a nucleic acid sequence encoding a gene that modifies the metabolic pathway of the secondary metabolite of interest and increases its expression;a terminator; a signal peptide; and optionally regulatory sequences that allow controlling the steps of transcription (e.g., polyA sequence) and / or translation.

[0055] In some embodiments, during step a), a molecular construct is introduced into bacteria before said bacteria are used to induce the formation of hairy roots in a plant, wherein said nucleic acid construct comprises at least one expression cassette, said expression cassette comprising: a promoter; a nucleic acid sequence encoding at least one gene that modifies the metabolic pathway of the secondary metabolite of interest and increases its expression; a terminator; optionally a signal peptide; and optionally regulatory sequences that allow controlling the steps of transcription (e.g., polyA sequence) and / or translation

[0056] In some embodiments, the promoter is a viral promoter, in particular a viral promoter derived from a Brassicaceae plant-infecting virus. In some embodiments, the promoter may be an inducible promoter, i.e., chemical or physical inducible system e.g., copper, steroid, alcohol, light), such as, for example, Tet repressor-based, tetracycline de-repressible; tTA-based, tetracycline inactivable; glucocorticoid receptor based, dexamethasone inducible; AlcR-based, ethanol inducible; Ecdysone receptor (EcR)-based, EcR agonist inducible; and estrogen receptor-based, P-estradiol inducible. In practice, a suitable promoter may be a constitutive Cauliflower Mosaic Virus (CaMV) 35S simple or double promoter or the Nos promoter.

[0057] In some embodiments, the expression cassette comprises regulatory sequences. In some embodiments the regulatory sequence is selected from the group consisting of a TMV enhancer, consensus sequence, or transcriptional factor.

[0058] In certain embodiments, the regulatory sequence may be a TMV enhancer.

[0059] In some embodiments, the expression cassette may comprise a polyadenylation signal that consists of multiple adenosine monophosphates. In practice, the expression cassette may also comprise a CaMV polyA sequence.

[0060] In some embodiments, the terminator sequence comprises a sequence from Agrobacterium tumefaciens (i.e., T-nos, tmas, toes, tORF25, ttml, tg7), from Solarium tuberosum (i.e., tpinll), from P is urn sativum (i.e., tE9) or from Glycine max i.e., t7S). In certain embodiments, a suitable regulatory sequence may be a CaMV T35S terminator.

[0061] In some embodiments, the expression cassette is then cloned into an expression vector, such as, e.g., a plasmid. Typically, the expression vector may be a binary vector suitable for expression in a plant cell, such as the pRD400, pBIN19, pBINPlus or pCAMBIA binary vector.

[0062] In practice, pBIN19, pBINPlus and pCAMBIA binary vector may be commercially available from Addgene®.

[0063] In some embodiments, the plasmid is a pRD400 plasmid.

[0064] In some embodiments, the plasmid may be selected in the group comprising pRD400 plasmid, pRLT plasmid and pSAMA plasmid.

[0065] Within the invention, the expression vector, e.g., the plasmid, may be incorporated into a competent bacterium by any one of the different processes known from the state of the art, such as bacterial transformation or electroporation.

[0066] As used herein, the term “competent” refers to a bacterium that has an increased ability to uptake an extra genomic nucleic acid into its cytoplasm. The skilled artisan is familiar with techniques for preparing competent bacteria (see, e.g., J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001)).

[0067] In some embodiments, competent bacteria for bacterial transformation may be chemically competent cells, in particular calcium chloride treated bacteria.

[0068] Electroporation consists in the utilization of an electrical field in a solution comprising plasmids and bacteria in order to increase the permeability of the bacteria cell membrane, allowing plasmids to be introduced in the bacteria. Suitable protocols may be found, e.g., in Green and Sambrook (Molecular Cloning, 4thEdition, 2012, Cold Spring Harbor Laboratory Press).

[0069] Typically, infection of the hairy roots, by a bacterium may be performed by contacting the bacterium with the hairy roots which have been wounded beforehand, as previously described in the state of the art.

[0070] In some embodiments, the bacterium may be Rhiwbium rhizogenes (formerly known as Agrobacterium rhizogenes), Rhizobium radiobacter (formerly known as Agrobacterium tumefaciens), or Rhizobium vitits (formerly known as Agrobacterium vitis). In practice, the bacterium used to infect the roots, in particular the hairy roots, may be Rhizobium rhizogenes (formerly known as Agrobacterium rhizogenes).

[0071] Many strains of Rhizobium rhizogenes (formerly known as Agrobacterium rhizogenes) can be used to perform the invention. Suitable strains include but are not limited to strain TR7 (or ATCC 25818 or LBA 9402), A4T, A4, ATCC 11325, LMG 155, LBA1334 and ATCC 15834.

[0072] In some embodiments, the strain of Rhizobium rhizogenes may be strain TR7, strain A4 or strain ATCC 15834.

[0073] In practice, the bacterium may be Rhizobium radiobacter (formerly known as Agrobacterium tumefaciens) harboring the rol genes, genetically integrated. Suitable strains include but are not limited to strain C58, C58C1, LBA4404, GV2260, GV3100, A136, GV3101, GV3850, EHA101, EHA105 and AGL-1.

[0074] In some embodiments, the strain of Rhizobium radiobacter may be strain GV3101 or strain AGL-1, preferably strain GV3101.

[0075] The “rol genes” refers to the group of bacterial genes capable of inducing the formation of hairy roots and also able to affect growth and morphogenetic potential of plant cells, at least in part by altering the capability to respond to plant hormones. In someembodiments, the hairy roots-based expression system, achieves the production of a secondary metabolite of interest.

[0076] According to the invention, the hairy roots-based expression system is a system wherein roots are genetically modified and are used to produce a secondary metabolite of interest.

[0077] In some embodiments, a molecular construct comprising one or more expression cassettes has been evaluated for its ability to produce the secondary metabolite of interest in high yield. This molecular construct is characterized by the use of a 35S double promoter, a TMV > enhancer, a PME signal peptide, the nucleic acid encoding at least one gene involved in a plant metabolic pathway and / or able to modify it, and a 35S terminator.

[0078] In some embodiments, said at least one gene involved in a plant metabolic pathway is encoding for enzymes and / or a transcription factor.

[0079] In some embodiments, the whole sequence is codon-usage optimized for Brassica rapa rapa taking into account a GC content around 50-60%. The sequence is then gene- synthesized and cloned into, first, an intermediary plasmid (pUC plasmid), then into at least one binary plasmid pRD400. The sequencing of the at least one pRD400 plasmid having integrated the molecular construct or, alternatively several molecular constructs, makes it possible to validate the integrity of the molecular construct. The at least one binary plasmid is then incorporated into competent R. rhizogenes bacteria by electroporation. Finally, the incorporation of the plasmid into the transformed R. rhizogenes clone is validated by DNA sequencing.

[0080] Plantlets of Brassica rapa rapa are then infected with this recombinant R. rhizogenes clone. The resulting clones are individualized and are all cultured in solid, then liquid culture medium. Antibiotics are only used for about 5 first cycles of culture and are only dedicated to eliminate R. rhizogenes. Apart from this very first step, all the process is antibiotic-free.

[0081] The first selection of the hairy root clones is based on their growth capacity. RNA is extracted from some of these hairy root clones and the integration of the gene (or genes) encoding the protein of interest, able to modify the metabolic pathway of the metabolite of interest and induce the overexpression of the secondary metabolite of interest, is confirmed by RT-PCR.

[0082] In some embodiments, the hairy roots-based expression system is able to produce the secondary metabolite of interest following a genetic modification of the hairy roots (i.e. said secondary metabolite of interest is produced by the genetically engineering hairy roots system). Preferably, the genetic modification encodes a protein allowing to modify the synthetic pathway of the secondary metabolite of interest.

[0083] In some embodiments, the secreted secondary metabolite of interest is selected in the group consisting of: polyphenols, alkaloids such as terpenoid indoles, cannabinoids, terpenoids, saponins, steroids, flavonoids, and tannins.

[0084] In some embodiments, the secreted secondary metabolite of interest is selected from the group consisting of ajmalicine, catharanthine, serpentine, tabersonine, vincristine, vindoline and vinblastine.

[0085] In some embodiments, the secreted secondary metabolite of interest is selected from the group consisting of ajmalicine, catharanthine and serpentine.

[0086] In some embodiments, the hairy roots-based system comprises a phase of culture - also called step of production - wherein the secondary metabolite of interest is produced by the hairy roots in a culture medium under conditions enabling the production of the secondary metabolite of interest. In other words, the method first comprises a phase of culture of hairy roots, in a culture medium, wherein the secondary metabolite of interest is produced and secreted by the hairy roots; under conditions enabling the production of the secondary metabolite of interest.

[0087] In some embodiments, the culture medium used in the step (a) of culturing the genetically engineered hairy roots, is suitable for rhizocal induction, thus the followingsteps of the method of the invention are conducted in rhizocals or in a culture comprising a mix of hairy roots and rhizocals.

[0088] In some embodiments, the method is performed in a sterile environment. In practice, the vessels and / or the culture media may be sterilized according to the protocols known from the state of the art. Examples of sterilization treatments include heattreatment (steam sterilization, high-temperature dry sterilization), UV treatment (UV-A, UV-B and / or UV-C), preferably UV-A, and gamma ray treatment.

[0089] In some embodiments, the culture of the hairy root-based system is performed in a suitable recipient.

[0090] In some embodiments, the recipient is a bioreactor or an Erlenmeyer flask. In some embodiments the bioreactor is at least a 25 L, at least a 200 L bioreactor, at least a 350 L bioreactor, at least a 500 L bioreactor, at least a 1000 L bioreactor or at least a 5000 L bioreactor.

[0091] In some embodiments, the phase of culture of roots is performed under bubbleoxygenation.

[0092] In some embodiments, a suitable culture medium used in the method according to the invention may comprise (i) one or more pH buffering system(s); (ii) one or more inorganic salt(s); (iii) one or more trace element(s); (iv) one or more free amino acid(s); (v) one or more vitamin(s); (vi) one or more hormone(s); (vii) one or more carbon / energy source(s).

[0093] Culture media for hairy roots-based expression systems are well known in the art. In some embodiments, a suitable medium, in particular for hairy roots-based expression systems, may be Standard Gamborg's (B5) medium, Murashige and Skoog's (MS) basal medium and N6 medium.

[0094] In some embodiments, the culture medium may comprise salt, preferably in a concentration inferior to 250 mM.

[0095] In some embodiments, during the step of culture of the genetically engineered hairy roots, culture growth may occur at a temperature ranging from about 15 °C to about 26°C, preferably from about 20°C to about 24°C and more preferably at 22°C or 23°C. In some embodiments, culture growth may occur at a temperature ranging from about 20°C to about 25°C.

[0096] In some embodiments, culture growth may occur in a light / dark photoperiod from about 13 h to about 18 h, preferably from about 15 h to about 17 h, and more preferably from about 16 h.

[0097] In some embodiments, the step a) of culture of the genetically engineered hairy roots is performed for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 days. It is understood that the duration of the step of culture may depend on the size of the recipient in which the culture is performed, as larger recipients may necessitate longer duration of the step of culture.

[0098] In some embodiments, the step (a) of culture is performed for about 7 days to about 45 days. Within the scope of the invention, the expression “for about 7 days to about 45 days” encompasses 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45 days.

[0099] In some embodiments, the step (a) of culture of the hairy roots is performed in a culture medium, wherein the culture medium is Gamborg B5 medium further comprising at least one saccharide. In some embodiments, the at least one saccharide is selected in the group comprising or consisting of sucrose, glucose, fructose, mannose, xylose and ribose. In certain embodiments, the saccharide is incorporated in the Gamborg medium at a concentration of from about 0.1% (0.1 g / 100 ml) to about 15% (15 g / 100 ml), preferably of from about 1% to about 5%, more preferably of about 3%.

[0100] In some embodiments, the culture medium may be Gamborg B5 medium with 3% sucrose.

[0101] In some embodiments, the culture medium may be renewed one or more times during the step a) of culture of the genetically engineered hairy roots, preferably by an identical culture medium, preferably by a volume identical to the initial volume.

[0102] In some embodiments, for the induction of rhizocals, an inductor of rhizocals is added to the culture medium after about 5 days to about 55 days of culture and preferably after about 14 days of culture. Within the scope of the invention, the expression “after about 5 days to about 55 days of culture” encompasses after about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and 55 days of culture.

[0103] In some embodiments, the induction of rhizocals is performed for about 5 days to about 30 days, preferably from about 10 to about 25, more preferably for about 14 days or about 25 days. In some embodiments, the phase of induction of rhizocals is performed for at least 5 days.

[0104] In some embodiments, the induction of rhizocals is performed in a culture medium in the presence of an inductor of rhizocals, in particular an auxin.

[0105] Within the scope of the invention “an inductor of rhizocals” means that the addition of said inductor of rhizocals in the culture medium leads to the appearance of rhizocals which are lateral root emergences appearing on hairy roots. These rhizocals are able to produce the compound of interest in a higher quantity than hairy roots with no rhizocals.

[0106] In some embodiments, the inductor of rhizocals is a hormone. In some embodiments, said hormone is an auxin.

[0107] In some embodiments, the auxin may be selected from the group comprising or consisting of 2,4-dichlorophenoxyacetic acid (2,4-D), 3-indoleacetic acid (IAA), indole -3-butyric acid (IBA), 1 -naphthaleneacetic acid (NAA),2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodoacetic acid,4-chlorophenoxy acetic acid, 2-naphthoxy acetic acid, 1 -naphthylacetic acid,4-amino-3,5,6-trichloropicolinic acid, 3,6-dichloro-2-methoxybenzoic acid (Dicamba), derivatives thereof and the likes. In some embodiments, the auxin is 2, 4-dichlorophenoxy acetic acid (2,4-D).

[0108] In some embodiments, the culture medium comprises an auxin, in particular 2.4- D, in a concentration of from about 0.1 mg / L to about 10 mg / L. Within the scope of the invention, “about 0.1 mg / L to about 10 mg / L” encompasses 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2,4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2,8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8 and 10.0 mg / L.

[0109] In some embodiments, the culture medium is Gamborg B5 medium with 3% sucrose and 1 mg / L of 2.4-D. The addition of an auxin, in particular 2.4-D, allows the cessation of the biomass growth simultaneously with the formation of rhizocals, leading to an increase of the ability of the roots to secrete the compound of interest. As used herein, the term “biomass”, as known in the art, refers to the total weight (amount) of living plants in a culture, expressed as weight per volume of culture (w / v). To measure the biomass, the roots are separated from the culture medium and they are weighted in an appropriate weighing scale and related to the initial culture volume. In some embodiments, the biomass may also be expressed as a dry biomass, the water contained in the roots being evaporated before weighing the roots. The dry biomass is expressed as a dry weight per volume of culture (dry w / v). In practice, evaporation of the water contained in the roots may be performed at a temperature of about 70 °C, for about 24 h.

[0110] In practice, the weight of the biomass is expressed in gram (g) or kilogram (kg), whereas the volume is expressed in milliliter (mL) or liter (L).

[0111] When the hairy roots-based system, in particular the hairy roots-based system, comprises an inducible promoter, the culture medium may comprise an effective amount of the corresponding inducer, such as, e.g., a tetracycline, a glucocorticoid (e.g., dexamethasone), an alcohol (e.g., ethanol), an estrogen e.g., [3-estradiol).

[0112] In some embodiments, the step (b) of elicitation consists in contacting the genetically engineered hairy roots to an elicitor into the culture medium so as to elicit theproduction of the secondary metabolite. In some embodiments, the elicitor can be a biotic elicitor, for example yeast extracts, fungal extract or living bacteria.

[0113] In some embodiments, the elicitor can be an abiotic elicitor, for example a component mimicking a stress, jasmonic acid, methyl jasmonate, salicylic acid or chitosan for signal molecules.

[0114] In some embodiments, an elicitor can be the application of a specific physical parameter such as temperature; light: for example, exposition of the roots to red light, green light, blue light, UV light : UV-A and UV-B, UV-C, preferably UV-B ; exposition to oxygen, or ozone. Examples of elicitors used in the method according to the invention include plant hormones, such as, e.g., auxins, and the like; phytopharmaceuticals, such as, e.g., methoxyfenozide, tubefenozide and the like; steroids, such as, e.g., dexamethasone, estradiol, and the like; alcohols, such as, e.g., ethanol, methanol, and the like; metal ions, such as, e.g., copper, silver, cadmium, cobalt, and the like; antibiotics such as, e.g., tetracycline; polyosides, such as, e.g., cyclodextrins, chitosan, chitin, sucrose, sorbitol, dextran, and the like; polypeptides, such as, e.g., elicitin; osmotic stress, osmotic pressure, salts and inorganic salts, such as, e.g., sodium orthovanadate, vanadyl sulphate, sodium chloride, and the like; organic salts, such as, e.g., methyl jasmonate, jasmonic acid, gibberellic acid, salicylic acid, sodium salicylate, abscisic acid and the like; proline; organic molecule, such as, e.g., 2,4-dichlorophenoxyacetic acid, polyethylene glycol, tween, PVP (polyvinylpyrrolidone), urea, and the like; yeast extracts; microorganisms, such as, e.g., Trichoderma atroviride, Protomyces gravidus, Claviceps purpurea, Mucor hivemalis, Fusarium oxysporum, Phoma exigua, Botrytis cinerea, Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli, Rhizobium leguminosarum.

[0115] In some preferred embodiments, in step (b), the elicitor is selected from the group consisting of: methyl jasmonate, jasmonic acid, chitosan, salicylic acid, jasmonate, cadmium chloride (CdC12), coumarine or furocoumarine, cyclodextrin, gibberellic acid, yeast extract, Phytopthora parasitica filtrate, Aspergillus niger, cellulase, and Bacteria sp.

[0116] In some preferred embodiments, in step (b) the elicitor is 2,4- dichlorophenoxyacetic acid.

[0117] In some embodiments, in step (b), the elicitor is an osmotic stress consisting in applying a salt to the culture medium, preferably at a concentration of 0.1 mM to 1 mM, or 1 mM to 10 mM, or 10 mM to 20 mM, or 20 mM to 30 mM, or 30 mM to 40 mM, or 40 mM to 50 mM, or 50 mM to 60 mM, or 70 mM to 80 mM, or 80 mM to 90 mM, or 90 mM to 100 mM, or 100 mM to 150 mM, or 150 mM to 200 mM, or 200 mM to 250 mM.

[0118] In some embodiments, in step (b), the elicitor is an osmotic stress consisting in applying a salt to the culture medium at a concentration of 0.1 mM to 10 mM, 0.1 mM to 25mM, 0.1 mM to 50mM, 0.1 mM to 100 mM, O.lmM to 150 mM, 0.1 mM to 200 mM, 0.1 mM to 250 mM.

[0119] In some embodiments, the elicitation stimulates the production of secondary metabolite, which leads to an accumulation of secondary metabolites inside the roots.

[0120] In some other embodiments, the elicitation stimulates the production of secondary metabolite, which leads to an accumulation of secondary metabolites inside the roots and to the secretion of some of the secondary metabolites in the culture medium.

[0121] In some embodiments, the step (b) of elicitation consists in contacting the genetically engineered hairy roots with an elicitor into the culture medium for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days. It is understood that the duration of this step of elicitation may depend on the size of the recipient in which the culture is performed, as larger recipients may necessitate longer duration of the step of elicitation, but also depend on the type of elicitor used.

[0122] In some embodiments, the phase of elicitation is performed for about 3 days to about 45 days. Within the scope of the invention, the expression “for about 3 days to about 45 days” encompasses 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45 days.

[0123] In some embodiments, the step (b) of elicitation is followed by a step (c) of induction of the secretion by addition of an agent, i.e. by addition of an agent which will promote the secretion of the secondary metabolites outside of the cells of the hairy root (or rhizocal). This process of secretion amplifies the amount of secondary metabolites in the culture medium.

[0124] In some embodiments, the step c) of induction of the secretion of the secondary metabolite is performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. It is understood that the duration of the step c) of induction of the secretion may depend on the size of the recipient in which the culture is performed, as larger recipients may necessitate longer duration of the step c) of induction of the secretion, but may also depend on the agent used for inducing the secretion.

[0125] In some embodiments, the step c) of induction of the secretion is performed for about 1 day to about 60 days. Within the scope of the invention, the expression “for about 1 day to about 60 days” encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 days.

[0126] In some embodiments, the method of the invention does not comprise any step in which the hairy roots are incubated in a solution comprising from about 250 mM to about 4M of a salt.

[0127] In some embodiments, the step c) of inducing the secretion of the secondary metabolite carried out by contacting the agent inducing the secretion with the genetically engineered hairy roots is performed under agitation.

[0128] Agitation refers to the action causing a slight movement of a solution / suspension in a recipient, preferably in a constant manner, in order to homogenize the distribution of the components in the solution / suspension.

[0129] In some embodiments, as known in the art, agitation may be performed by mechanic or magnetic means. In some embodiments, the agitation may be performed by placing the recipient which contains the hairy roots with the agent inducing the secretion on a constant moving support (e.g., a shaking table or an orbital shaker). Agitation performed by placing the recipient which contains the hairy roots with the agent on a constant moving support is also referred to as shaking. In some embodiments, the agitation is performed with the means of a magnet in the recipient containing the hairy roots with the agent inducing the secretion and a magnetic agitator on which the recipient is placed.

[0130] In practice, the agitation may be performed using mechanical means and more particularly by placing the recipient which contains the hairy roots with the agent inducing the secretion on a constant moving support, such as a shaking table or an orbital shaker.

[0131] In some embodiments, the agitation, in particular the shaking, is performed at a speed of from about 10 rpm to about 350 rpm, preferably at a speed of from about 10 rpm to about 300 rpm, more preferably at a speed of from about 10 rpm to about 100 rpm. Within the scope of the invention the expression “about 10 rpm to about 300 rpm” encompasses 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 and350 rpm. Within the scope of the invention the expression “about 10 rpm to about300 rpm” encompasses 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 and300 rpm. Within the scope of the invention the expression “about 10 rpm to about100 rpm” encompasses 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95 and 100 rpm.

[0132] In some preferred embodiments, the agitation is a gentle stirring. In some embodiments, a gentle stirring is a gentle shaking. In some embodiments, a gentle stirring is a gentle shaking performed at a speed of from about 10 rpm to about 100 rpm. In some embodiments, a gentle stirring is performed with no shaking and only by oxygenation of the culture, for example by bubble-oxygenation.

[0133] In some preferred embodiments, the step of contacting the hairy roots, with an agent inducing the secretion is performed without shaking.

[0134] In some embodiments, the agitation is performed by stirring (i.e. performed by mechanical means, for example with a pale).

[0135] In some preferred embodiments, the agitation is performed using an air-lift system in which the liquid is put into motion by generated air bubbles. An example of such system is the air-lift system described in the patent application PCT / EP2023 / 058535.

[0136] In some embodiments, the secretion of secondary metabolites out of the hairy roots, or rhizocals, is induced by the addition of a compound to the culture medium. Examples of such compounds capable of inducing the secretion of the secondary metabolites are ethanol, chitosan, salicylic acid, jasmonic acid, Tween20, pH (2<pH< 8).

[0137] In some embodiments, the step of (i) collection of the culture medium containing the secreted metabolite of interest and the (ii) addition of fresh culture medium are carried out one after the other.

[0138] In some embodiments, the step (i) of collection is carried out by any classical methods well known by the person skilled in the art, such as collecting the culture medium containing the secreted metabolite of interest.

[0139] The efficacy of collection of the secondary metabolite of interest may generally be measured by any suitable means for detecting said metabolite. Examples of suitable means for detecting a compound of interest may be ELISA, Western Blotting, immunoprecipitation, mass spectrometry, fluorescence, enzymatic assay, flow cytometry and the likes.

[0140] In some embodiments, the totality of the culture medium is collected in sterile conditions at step (i) and used as starting material for the downstream process. Fresh and sterile culture medium is then added at step (ii) to the culture in order to initiate the next secretion cycle of metabolite of interest.

[0141] In some embodiments, the totality of the culture medium is collected in sterile conditions at step (i) and fresh and sterile culture medium is then added at step (ii) to the culture.

[0142] In some embodiments, at least the steps c) and d) of the method according to the invention are repeated sequentially at least one time so as to begin consecutively a new cycle of collection without interruption of the production.

[0143] In some embodiments, at least the steps c) and d) are repeated at least one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten time, preferably one time, two times or three times.

[0144] In some embodiments, at least the steps c) and d) of the method can be repeated between 2 and 10 times, preferably between 2 and 8 times, even more preferably between 2 and 5 times.

[0145] In some embodiments, the steps b), c) and d) of the method according to the invention are repeated sequentially at least one time so as to begin consecutively a new cycle of collection without interruption of the production.

[0146] In some embodiments, the steps b), c) and d) are repeated at least one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, preferably one time, two times or three times.

[0147] In some embodiments, the steps b), c) and d) of the method can be repeated between 2 and 10 times, preferably between 2 and 8 times, even more preferably between 2 and 5 times.

[0148] In some embodiments, the step of collection (i) can be repeated so as to carry out several collection cycles.

[0149] In some embodiments, the step of collection (i) can be repeated every 1 day, or every 2 days, or every 3 days, or every 4 days, or every 5 days, or every 6 days, or every 7 days, or every 8 days, or every 9 days, or every 10 days, or every 11 days, or every 12 days, or every 13 days, or every 14 days, or every 15 days, or every 16 days, or every 17 days, or every 18 days, or every 19 days, or every 20 days, before adding the fresh culture medium and starting a further new cycle of production.

[0150] In some embodiments, the interval between two steps of collections (i), after renewal of the culture medium is less than 20 days, less than 10 days, preferably less than 8 days, even more preferably 5 days.

[0151] In some embodiments, the number of collection cycles that can be achieved with the method of the invention is between 2 and 10 times, preferably between 2 and 8 times, even more preferably between 2 and 5 times.

[0152] In some embodiments, the elicitation phase is repeated after a step of collection, and the following steps of collection and culture medium renewal are thereafter repeated at least one time, preferably at least twice, even more preferably at least three times.

[0153] In some embodiments, the steps b), c) and d) are repeated sequentially at least one time.

[0154] In some embodiments, only the steps c) and d) are repeated sequentially at least one time.

[0155] In some embodiments, the steps b) and c) are concomitant.

[0156] In some embodiments, the method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system comprises or consists of:(al) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction;(bl) a step of elicitation of the production of a secondary metabolite by applying an elicitor;(cl) a step of induction of the secretion of the secondary metabolite into the culture medium by addition of an agent; and(dl) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium; and(c2) optionally at least one additional step of induction of the secretion of the secondary metabolite by addition of an agent, preferably about at least two to ten; and(d2) at least one additional step, of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium, preferably about at least two to ten.

[0157] In some embodiments the method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system comprises or consists of:(al) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction;(bl) a step of elicitation of the production of a secondary metabolite by applying an elicitor;(cl) a step of induction of the secretion of the secondary metabolite into the culture medium by addition of an agent; and(dl) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium.

[0158] In some embodiments the method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system comprises or consists of:(al) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction;(bl) a step of elicitation of the production of a secondary metabolite by applying an elicitor;(cl) a step of induction of the secretion of the secondary metabolite into the culture medium by addition of an agent; and(dl) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium; and(d2) at least one additional step, of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium, preferably about at least two to ten.

[0159] In some embodiments the method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system comprises or consists of:(al) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction;(bl) a step of elicitation of the production of a secondary metabolite into the culture medium by applying an elicitor; and(dl) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium; and(d2) at least one additional step, of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium, preferably about at least two to ten.

[0160] In some embodiments, the method comprises a final step of purification of the secondary metabolite of interest. The person skilled in the art knows classical methods allowing to purify said secondary metabolite of interest in the context of the invention.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1 is a schematization of secondary metabolites production process by hairy roots.Figure 2 is a schematization of the cyclic production (Figure 2A) and the control experiment (Figure 2B). Fig.2A after 14 days of growth phase, the culture medium is renewed and the elicitor 2,4-D is added for a production phase of 7 days (cycle n°l). Onday 21, the culture medium is renewed and 2,4-D added for a production phase of 7 days (cycle n°2). On day 28, the culture medium is renewed and 2,4-D added for a production phase of 7 days (cycle n°3). Samples of the culture medium is collected on days 21, 28 and 35. Fig. 2B after 14 days of growth phase, the culture medium is renewed once but no elicitation is done. The culture medium is not renewed for a production phase of 21 days. Samples of the culture medium is collected on days 21, 28 and 35.Figure 3 is a combination of graphs showing the accumulation of TIAs in the culture medium of hairy roots in the context of cyclic production or in the control (without elicitation with 2,4D or medium renewal). The quantity of ajmalicine (Fig 3A), catharanthine (Fig. 3B) and serpentine (Fig. 3C) were measured on day 21 (end of cycle n°l), day 28 (end of cycle n°2) and day 35 (end of cycle n°3). Fig.3A The quantity of ajmalicine in the culture medium increased with every cycle of cyclic production whereas in the control the quantity of ajmalicine in the culture medium remained very low during the 35 days. Fig.3B The quantity of catharanthine in the culture medium increased with every cycle of cyclic production whereas in the control the quantity of catharanthine in the culture medium remained very low during the 35 days. Fig.3C The quantity of serpentine in the culture medium increased with every cycle of cyclic production whereas in the control the quantity of serpentine in the culture medium remained very low during the 35 days. The quantity is expressed in MS / MS peak integration (AU)EXAMPLESExample 1: Cyclic production of terpenoid indole alkaloids (TIA) in hairy root clones of Catharanthus roseus (C. roseus)Catharanthus roseus is a medicinal plant that produces alkaloids. These alkaloids are located in various parts of the plant, for example catharanthine is found in the leaves, stems and roots while ajmalicine is found in the stems and roots of Catharanthus roseus.Material and methods1. Molecular constructs and generation of modified hairy rootsHairy root clones of Catharanthus roseus were modified to optimize the production of TIA by a step of genetic engineering. Two molecular constructs were designed to overexpress the genes encoding for 4 keys enzymes: strictosidine P-D-glucosidase (SGD), tryptophan decarboxylase (TDC), geraniol 10-hydroxylase (G10H), strictosidine synthase (STR) and one transcription factor (ORC A3) involved in the TIA biosynthesis. Plasmid 1 encodes genes that overexpress TDC, STR and G10H ; and plasmid 2 encodes genes that overexpress ORCA3 and SGD. Hairy root clones were infected with R. rhizogenes in the presence of the two plasmids. Three clones were used for each condition tested.The clones were maintained in shake flasks in 100 mL of * B5 Gamborg medium supplemented with 30g / L of sucrose at 25 °C and under agitation (100 rpm).2. Quantification of TIA in the modified hairy roots and wildtype hairy roots2. a. Extraction of TIA from the biomass3g of biomass was harvested from each hairy root clone, for the quantification of secondary metabolites inside the hairy root, specifically of TIA. The samples were lyophilized, then grinded using the Geno Grinder device 2x30s at 1200 rpm with 1 bead of 10mm. 30 mg of the freeze-dried biomass (done in triplicate) were weighed and mixed with 1,2 mL of 100% methanol. The samples were then shaken using magnetic bar at 500 rpm during one hour and centrifuged at 12,000xg for five minutes. The supernatant was harvested and 90pL of this “liquid biomass” was mixed with 810pL of 100% methanol in vials.2. b. Extraction of TIA from the culture mediumDuring the cyclic production, the continuous production, and the controls, at the end of each cycle, the culture medium was harvested. 450pL of the collected culture medium was mixed with 450 pL of 100% Methanol in vials for quantification of TIA.The quantification was done using the Acquity H-Class UHPLC coupled to Vion (HRMS) (Waters ™). The conditions for the liquid chromatography were:Column: Kinetex 1.7pm Biphenyl 100 A, 100x2.1 mm (S / N H23-329201, B / N 5628-0050) (Phenomenex ™)- Gradient : A (H2O+0.1%AF), B (MeOH+0.1%AF), %A (time, min) = 80 (0), 80 (0.5), 10 (6.0), 10 (7.0), 80 (7.5), 80 (10).Flow rate: 0.5 mL.min-1Column temperature: 55°CSample temperature: 15°CInjected volume: 0.5 pLThe data were processed using the Unifi software (Waters ™).The conditions used for the mass spectrometer were :Polarity : ESI+Tension : 3 kVSampling cone : 40 VSource Offset : 80 VSource Temperature : 120°CDesolvation Temperature : 450°CCone Gas Flow : 50 L / hDesolvation Gas Flow : 1000 L / hMass range : 50 - 2000 DaMS chain collision energy: 6 eVMSEchain collision energy: gradient 25 - 50 eVThe modified hairy roots of C. roseus contained in their biomass high levels of three TIAs (ajmalicine, catharanthine and serpentine). However, extracting these TIAs from the biomass requires destroying the hairy roots. The aim of the cyclic production is tohave high quantities of secondary metabolites produced by the hairy root and released in the culture medium, which can be easily collected without damaging the hairy root.1. Step of genetic engineeringThe modification of hairy roots was done as described in Material and Methods 1.2. Step of biomass growthIn order to evaluate the ability of the hairy root clones to produce the molecules of interest in successive cycles, 60mg / 6 mL, two sets of triplicates of the hairy root clone 151 were seeded using Yi B5 medium supplemented with 30g / L of sucrose, and left to grow for 14 days at 25°C under 70 rpm agitation. (Fig. 3A)3. Step of elicitationOn day 14, the culture medium was renewed using Yi B5 medium supplemented with 30g / L of sucrose and 1 mg / L of 2,4 dichlorophenoxyacetic acid to increase the production of TIA while reinforcing the key enzymes activity. This first cycle of production of secondary metabolites lasted 7 days. (Fig. 3A)4. Repetition of the step of elicitation and renewal of the culture mediumOn day 21, the culture medium was renewed using Yi B5 medium supplemented with 30g / L of sucrose and 1 mg / L of 2,4 dichlorophenoxyacetic acid. This second cycle of production of secondary metabolites lasted 7 days. (Fig. 3A)On day 28, the culture medium was renewed using Yi B5 medium supplemented with 30g / L of sucrose and 1 mg / L of 2,4 dichlorophenoxyacetic acid. This third cycle of production of secondary metabolites lasted 7 days. (Fig. 3A)5. Control groupFor the control group, steps 1 and step 2 were identical to the cyclic production group.However, there was no step of elicitation and no step of renewal of the medium.On day 14, the culture medium was replaced by Vi B5 medium supplemented with 30g / L of sucrose but no 2,4 dichlorophenoxyacetic acid. No renewal of the culture medium was done during 21 days.6. Collection of culture medium and biomass for the quantification of the secondary metabolitesOn days 21, 28 and 35, samples of the culture medium were collected from both the cyclic production hairy roots and from the control hairy roots. The samples were prepared (as described in Material and Methods 2.b) and the TIA were quantified using liquid chromatography coupled with mass spectrometry (as describedin Material and Methods 2.c).ResultsThe quantity of ajmalicine in the culture medium increased from 25192 AU at the end of cycle 1, to 83532 AU at the end of cycle 2 and 102463 AU at the end of cycle 3. In the control hairy-root there was no significant increase of the quantity of ajmalicine in the culture medium (6381 AU on day 21, 5639 AU on day 28 and 14840 AUon day 35). (Fig.3A)The quantity of catharanthine in the culture medium increased from 4426 AU at the end of cycle 1, to 27078 AU at the end of cycle 2 and 51593 AU at the end of cycle 3. In the control hairy -root there was no significant increase of the quantity of catharanthine in the culture medium (0 AU on day 21, 1032 AU on day 28 and 2745 AU on day 35). (Fig. 3B)The quantity of serpentine in the culture medium increased from 7662 AU at the end of cycle 1, to 32570 AU at the end of cycle 2 and 185996 AU at the end of cycle 3. In the control hairy-root there was no significant increase of the quantity of serpentine in the culture medium (2080 AU on day 21, 2233 AU on day 28 and 5364 AU on day 35). (Fig. 3C)The quantity of the three TIA : ajmalicine, catharanthine and serpentine, released in the culture medium, increased in each culture cycle with the cyclic production. Indeed, thehairy roots, when placed in the conditions of the cyclic production, can produce secondary metabolites for at least three production cycles.In the absence of renewal of the culture medium + 2,4D, only very low levels of TIA were detected in the culture medium (Fig.3ABC). In the absence of elicitation and renewal of the culture medium, the secondary metabolites expressed by the hairy root remain inside the plant. Thus, the cyclic production allows the recovery of these metabolites without having to destroy the plant.

Claims

CLAIMS1. A method for the continuous production of a secreted secondary metabolite of interest by a hairy roots-based expression system, the method comprising: a) a step of culture of genetically engineered hairy roots in a culture medium, optionally in a culture medium suitable for rhizocal induction; b) a step of elicitation of the production of a secondary metabolite of interest by applying an elicitor; c) optionally a step of induction of the secretion of the secondary metabolite into the culture medium by addition of an agent; and d) a step of (i) collection of the culture medium containing the secreted metabolite of interest and (ii) addition of fresh culture medium, wherein at least said steps c) and d) are repeated sequentially at least one time.

2. The method according to claim 1, wherein the steps b), c) and d) are repeated sequentially at least one time.

3. The method according to claims 1 or 2, wherein the method does not comprise any step in which the hairy roots are incubated in a solution comprising from about 250 mM to about 4M of a salt.

4. The method according to any one of claims 1 to 3, wherein the secreted secondary metabolite is selected in the group consisting of: polyphenols, alkaloids, cannabinoids, terpenoids, saponins, steroids, flavonoids, and tannins.

5. The method according to any one of claims 1 to 4, wherein the genetically engineered hairy roots are obtained by introducing a nucleic acid encoding for at least one gene involved in a plant metabolic pathway.

6. The method according to any one of claims 1 to 5, wherein the elicitor is selected from the group comprising or consisting of : plant hormones, such as, e.g., auxins, and the like; phytopharmaceuticals, such as, e.g., methoxyfenozide, tubefenozideand the like; steroids, such as, e.g., dexamethasone, estradiol, and the like; alcohols, such as, e.g., ethanol, methanol, and the like; metal ions, such as, e.g., copper, silver, cadmium, cobalt, and the like; antibiotics such as, e.g., tetracycline; polyosides, such as, e.g., cyclodextrins, chitosan, chitin, sucrose, sorbitol, dextran, and the like; polypeptides, such as, e.g., elicitin; inorganic salts, such as, e.g., sodium orthovanadate, vanadyl sulphate, sodium chloride, and the like; organic salts, such as, e.g., methyl jasmonate, jasmonic acid, gibberellic acid, salicylic acid, sodium salicylate, abscisic acid and the like; proline; organic molecule, such as, e.g., 2,4-dichlorophenoxyacetic acid, polyethylene glycol, tween, PVP (polyvinylpyrrolidone), urea, and the like; yeast extracts; microorganisms, such as, e.g., Trichoderma atroviride, Protomyces gravidus, Claviceps purpurea, Mucor hivemalis, Fusarium oxysporum, Phoma exigua, Botrytis cinerea, Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli, Rhizobium leguminosarum; red light, green light, blue light, temperature, oxygen, pH, ozone, UV-C, osmotic stress, and combinations thereof.

7. The method according to any one of claims 1 to 6, wherein the elicitor is selected from the group consisting of: methyl jasmonate, jasmonic acid, chitosan, salicylic acid, jasmonate, cadmium chloride (CdC12), coumarine or furocoumarine, cyclodextrin, gibberellic acid, yeast extract, Phytopthora parasitica filtrate, Aspergillus niger, cellulase, 2,4-dichlorophenoxyacetic acid and Bacteria sp.

8. The method according to any one of claims 1 to 7, wherein the elicitor is an abiotic elicitor selected from the group consisting of red light, green light, blue light, temperature, oxygen, pH, ozone, UV-C, osmotic stress, and combinations thereof.

9. The method according to any one of claims 1 to 8, wherein the elicitor is an exposition of the hairy roots to UV, preferably UV-A and / or UV-B.

10. The method according to any one of claims 1 to 9, wherein the hairy roots are incubated in the culture medium with the elicitor at step b) for about 3 days to about 30 days, preferably for about 3 days to about 20 days.

11. The method according to any one of claims 1 to 10, wherein the secretion is induced by ethanol, chitosan, salicylic acid, jasmonic acid, Tween20, pH (2<pH< 8).

12. The method according to any one of claims 1 to 11, wherein the secretion lasts for about 1 day to about 60 days, preferably for about 1 day to about 20 days.

13. The method according to any one of the preceding claims, wherein said hairy roots belong to the species Brassica rapa rapa, Brassica napus, Salvia milthiorrhiza, Panax ginseng, Armoracia rusticana, Trigonella foenumgraceum, Lippia dulcis, Lithospermum erythrorhizon, Ophiorrhiza pumila, and. Echinacea purpurea, Echinacea angustifolia, Puerariaphaseoloides, Harpagophytum procumbens, Morinda citrifolia, Hypericum perforatum, Derris trifolia, Salvia miltiorrhiza, Salvia prevalzkii, Echinacea pallida, Cistanche tubulosa, Glycyrrhiza glabra, Sophoraflavescens, Rhodiola rosea, Polygonum cuspidatum, Fallopia multiflora, Lepidium peruvianum, Whitania Somnifera, Astragalus Membranaceous, Berberis Vulgaris, Sanguinaria canadensis, Eleutherococcus senticosus, Cannabis sativa, Hydrastis canadensis, Arctium majus, Piper methysticium, Pueraria lobata, Glycyrrhiza uralensis, Ptychopetalum olacoides, Dioscorea vollosa, Yucca shidigera, Panax quinque folius, Azadirachta indica, Catharanthus trichophyllus, Calystegia sepium, Atropa belladonna, Hyoscyamus muticus, Duboisia myoporoides, Duboisia leichhardtii, Artemisia annua, Datura stramonium, Arabidopsis thaliana, Stizolobium, Hassjoo, Ipomea aquatica, Perilla fruitescnens, Catharanthus roseus, Taxus brevifolia, Gloriosa superba, Saponaria officinalis, Solanum tuberosum, Nicotiana tabacum, Nicotiana benthamiana or Cinchosa pubescens, preferably to the Brassica rapa or Brassica napus species.

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