EP1747278A1 - Methode de production de peptides cycliques a partir de cultures cellulaires vegetales in vitro - Google Patents

Methode de production de peptides cycliques a partir de cultures cellulaires vegetales in vitro

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Publication number
EP1747278A1
EP1747278A1 EP05739387A EP05739387A EP1747278A1 EP 1747278 A1 EP1747278 A1 EP 1747278A1 EP 05739387 A EP05739387 A EP 05739387A EP 05739387 A EP05739387 A EP 05739387A EP 1747278 A1 EP1747278 A1 EP 1747278A1
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EP
European Patent Office
Prior art keywords
cell
species
culture
cyclic peptides
proteins
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EP05739387A
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German (de)
English (en)
Inventor
Heike DÖRNENBURG
Peter Seydel
Rainer Buchholz
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BUCHHOLZ, RAINER
DOERNENBURG, HEIKE
SEYDEL, PETER
Original Assignee
Friedrich Alexander Univeritaet Erlangen Nuernberg FAU
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Application filed by Friedrich Alexander Univeritaet Erlangen Nuernberg FAU filed Critical Friedrich Alexander Univeritaet Erlangen Nuernberg FAU
Publication of EP1747278A1 publication Critical patent/EP1747278A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present invention relates to a method for producing cyclic peptides, polypeptides or proteins having a cyclic cysteine knot or its chemical or structural equivalent in an in vitro culture system of plant origin. Further, the present invention relates to a device for producing these cyclic peptides, polypeptides or proteins.
  • Plant tissue cultures and plant cell cultures are used as biocatalysts for transformation of substrates by single enzyme reactions or multi enzyme steps (biotransformations) and de novo syntheses.
  • a successful example for the application of cell culture technology using higher plants in a production process is the production of Taxol (WO 97/44476) .
  • the induced metabolite delivery due to permeabilisation of cell membranes and/or the accumulation of the secondary metabolites formed by in situ product recovery or perfusion techniques is well known in the art.
  • a prerequisite for the commercial use of plant cell cultures is the ability to develop systems like cell reactors with immobilised cells, which enable an efficient use of the biomass produced.
  • the immobilisation of the plant cells offers a number of advantages in the production secondary plant compounds.
  • a further important issue in these systems is that the continuous release of the mostly intracellularly accumulated substances is initiated in the respective nutrition or production media. Therefore, a high product yield is obtained without the disadvantages of destroying the cell culture. Representative examples for these strategies are published by D ⁇ rnenburg and Knorr, Proc. Biochem. 27 (3 ), 161-166, 1992 or D ⁇ rnenburg and Knorr, Enzyme Microb. Technol . 17(8), 1995, 674-684.
  • the continuous removal of produced metabolites from the cultivation medium after addition of an extra cellular accumulation site shall avoid problems due to product inhibition and/or degradation.
  • Proteins have been traditionally regarded as linear chains of amino acids which fold into a defined three-dimensional shape necessary to enable their biological function.
  • the linear peptide backbone is cross-linked via disulfide bonds between cysteine residues.
  • the three dimensional folds are topologically simple and are not knotted.
  • Certain plants of the Rubiaceae, Violaceae and Curcubitaceae families provide small cyclic proteins in the order of approximately 30 amino acids. The cyclization involves an amide bond resulting in no identifiable N-or C- terminus in the molecule.
  • cyclic molecules are the circulins (Gustafson et al, 1994), Kalata Bl (Saether et al, 1995), cyclopsychotride (Witherup et al, 1994) and several molecules from the Violaceae family (Schopke et al, 1993; Claeson et al, 1998; Goransson et al, 1999) .
  • the objective problem underlying the invention was therefore to provide a new method for producing said cyclic peptides, polypeptides or proteins on an industrial scale.
  • the cysteine knot involves two intracysteine backbone segments and their connecting disulfide bonds, CysI-CysIV and CysII-CysV, which form a ring that is penetrated by the third disulfide bond, CysIII-CysVI .
  • said cyclic peptides, polypeptides or proteins comprise a beta-hairpin structure, other preferred structures are moebius and non-moebius structure .
  • said cyclic peptides comprise amino acids in a range from about 20 to about 100 more preferred in a range from about 20 to about 40 or still more preferred in a range from 29 to 35.
  • the method according to the invention is particularly useful for the production of circulins, cycloviolins, cycloviolacin, kalatas, cyclopsychotrids, palicoureins, viola peptides like for example vitri, vodo, vico, varv peptides, hypa A or McoTO .
  • a very important factor in performing the method according to the invention is the influence of electromagnetic irradiation, i.e. light in the UV/VIS range.
  • a typical irradiation protocol means for example an irradiation over a period of 16 hours followed by 8 hours without irradiation and exclusion of light in the bioreactor, where the method according to the invention is carried out.
  • the energy of the light source is in the range of 10 to 100 ⁇ Em "2 S _1 , more preferred from 20 to 15 ⁇ Em ⁇ S -1 , and most preferred from 25 to 35 ⁇ Em "2 S _1 . In the case of Violaceae species this value is in the range of 30 ⁇ Eirf 2 S -1 .
  • irradiation sources with different wavelengths and energy can be used within the scope of the present invention. It is well-known to an artisan, to perform experiments without undue burden and with various irradiation sources without departing from the scope of the invention. In less preferred embodiments, there are cell lines from the below mentioned plant species, can be established without the influence of light, for example by adding vitamins or phytohormones .
  • the plant cell or tissue culture includes but is not limited to Chassalia species, Oldenlandia species, Psychotria species, Palicourea species, Leonia species, Hybanthus species, or Momordica species .
  • the plant cell or tissue culture may be recombinant or not recombinant .
  • Typical processes to obtain recombinant cell cultures are indirect and direct DNA transfer, transformation via A. tumefaciens (shooty teratomas) and A. rhizogenes (hairy roots) , transformation via vectors (artificially constructed) and transfection via viruses, gene gun, particle gun, microinjection, electroporation, chemical methods, e.g. with polyethylenglycol, naked DNA (packaged in liposomes and spheroplasts) .
  • Suitable plant materials are protoplasts, cell cultures or plants
  • the cell culture system may be a differentiated one like hairy roots or an non- differentiated cell culture system.
  • Examples for undifferentiated cell culture systems are for example Callus cell cultures, fine suspension cultures, and meristemic tissue.
  • morphologically undifferentiated cells for the production of useful metabolites in ways similar to microorganisms and there are many examples which show high productive ability of such cells compared with intact plants.
  • the development of a certain level of differentiation is considered to be important in the successful production of phytochemicals by cell cultures.
  • Differentiated cell cultures are characterised by the ability of cell division and regeneration of new tissue.
  • organic cultures are cultures of differentiated tissue, that originate from isolated organs or organ parts, like roots or stems of plants, which are organised and grow differentiated.
  • callus cultures can be obtained on solid nutrition medium as surface cultures and in liquid nutrition medium as aggregate or fine suspension cultures .
  • Preferred examples for differentiated cultures are suspension cultures, embryonic cell cultures, root cultures, shooty teratomas, hairy roots.
  • the metabolism of plant cells strongly depends on the aggregation and the differentiation state of the cultures.
  • the original differentiation is achieved by the influence of neighbour cells and tissues.
  • the cell-to cell-contact is therefore also a decisive factor for biochemical differentiation .
  • Differentiated cell cultures have also a higher biochemical and genetic stability than non organised cultures (Parr, A.J., J. Biotechnol . 10, 1989, 1-26).
  • non differentiated cell cultures are not able to synthesise secondary metabolism products, which are therefore less preferred for the purpose of the present invention. Only after organic and thereby also after biochemical differentiation, they do so. That is due to a positive correlation between the stability of secondary metabolism syntheses and the organisation and differentiation of plant cells .
  • the plant cell or tissue culture is usually cultivated by a batch, fed-batch, repeated fed-batch or in a continuous perfusion modus process.
  • the expert skilled in the art is familiar with the advantages and disadvantages of these routine methods .
  • the most preferred method for the production of the peptides, polypeptides or proteins according to the invention comprises the induction of the production by changing the composition of the nutrient medium.
  • the change in the composition and therefore the induction of the expression of one or more of the cyclic peptides, polypeptides or proteins according to the invention is carried out in especially preferred embodiments by adding an elicitor, a precursor or an elicitor and a precursor to the nutrient medium.
  • elicitor in the context of the present invention means compounds and factors which trigger the synthesis and accumulation of plant compounds. They are classified in biotic (biological) and abiotic (chemical and physical) elicitors. An elicitor acts as an endogeneous or exogenous elicitor.
  • elicitors can be added in a continuous mode, in a pulsed mode or during the sensitive phases of the plant development .
  • the elicitation of plant cell cultures by the addition of elicitors takes place at concentrations which are dependent on the specific elicitor. Some elicitors are in specific concentrations toxic for the plant cell cultures. For oligosaccharides, the preferred concentrations range from 10 to 500 ⁇ g/ml. Jasmonic derivatives are added in a range from 10 to 200 ⁇ g/ml during sensitive growth phases (dependent on the cell culture) , at fast growing cultures approximately 2 to 3 days, in slower growing cultures (e.g. embryonic cultures) after 7 to 11 days.
  • the addition of elicitors is preferably carried out in a pulsed protocol during the growth phase of the plant cell culture and/or in the stationary phase (for incubation times see below) .
  • the induction of stress by elicitors during the acceleration phase of a plant culture is achieved e.g. in an Oldenlandia fine suspension 2 to 3 days after inoculation, in a slow growing culture, e.g. a Viola tricolor embryonic culture after 7 to 11 days.
  • the elicitor is added in a discontinuous protocol, it will be added for example to an Oldenlandia affinis fine suspension culture after 3 to 4 days in the growing culture, or after 7 days in a stationary phase.
  • Values for corresponding Viola tricolor embryogenic culture are 14 to 17 days (growing culture) and 25 to 28 days (stationary phase) .
  • Chemical elicitors comprise for example metal compounds like HgCl 2 , CuS0 4 , inhibitors of protein syntheses like actinomycine D or cycloheximide, respiratory inhibitors like CN “ or 2 , 4-dinitrophenol, pesticides and detergents.
  • Further suitable chemical elicitors for the use in the present invention are ozone, herbicides, e.g. acifluorfen, alpha-amino butyric acid, nitric oxide, sodium nitroprusside, silicium oxide.
  • Further physical elicitors are for example temperature impulses like cold shock, UV radiation for the influence of light, electroporation or the treatment with high hydrostatic pressures.
  • Biological elicitors comprise for example cell wall parts from fungi and bacteria, enzymes and other metabolites, plant oligosaccharides which are released by injury or infection from the plant cell wall and viruses which induce enzyme activities by infection.
  • Preferred elicitors in the context of the present invention are jasmonic acid derivatives, oxylipins, fatty acids, oligo- and polysaccharides from microorganisms or algae, and oligo galacturonic acids.
  • Non-limiting examples of polysaccharides as elicitors originating from plants are: guar gum, CM-cellulose, pectic acid, pectin, konjac, cellulose, ⁇ -cyclodextrin, locust beam gum, alginate and from microorganisms: alginate, rhamsan, xanthan, chitin, chitosan, curdlan, levan, gellan, welan.
  • Microbial polysaccharides like chitosan, are naturally occurring components in the cell wall of numerous fungi, which can be obtained by the treatment of chitin with a hot calcium hydroxide solution.
  • Chitosan is a partially deacetylated polymeric ⁇ -1 , 4-N-acetyl-D-glucosamine and has variable positive charges depending on the degree of acetylation.
  • precursors or related compounds to the culture media further stimulates secondary metabolite production. This approach is especially advantageous if the precursors are inexpensive.
  • precursors for biotransformation are substances from chemical syntheses but also from biological origin, either from plant cell cultures of the same or a different species or from microorganism.
  • Preferred precursors are primary metabolites.
  • sulphur containing amino acids are especially preferred as well as reductive components as for example glutathione.
  • Precursors are usually added with the transfer of the culture (at day zero) during growth phases or after termination of exponential growth. Preferred concentrations are 0,1 to 10 mMol .
  • precursors are also added during the addition of elicitors to support cells in the production of the cyclic peptides.
  • precursors are added from the very beginning in the medium in continuous protocol, since for example the reduced precursors are sometimes not stable.
  • cyclic peptides For the production of cyclic peptides according to the invention, a large number of precursors can be used. Preferred are amino acids, which are used for the generation of the active peptides. L-cysteine is an especially preferred precursor amino acid, since cysteine dimerises to cystine by a disulfide bridge formation which leads to the stability of the cyclic peptides of the plants. Preferred concentrations for precursors in the context of the present invention are usually between 1 to 10 mM of the precursor.
  • a further preferred embodiment according to the invention is the initiation of organ cultures (as shoot or root cultures) by tumour formation with agrobacteria for the production of the cyclic peptides.
  • the plant cell or plant tissue culture is immobilised.
  • Immobilisation means the fixation of the cells in or absorbed on the particles and the incorporation of biocatalysts in a reactor (for example membrane reactor) wherein the product release and continuous use of biocatalysts is reported.
  • Immobilisation further improves the volumetric productivity of secondary metabolites.
  • the immobilisation supports a continuous process; the biocatalyst can be used more often and is easier to separate from the cultivation medium.
  • plant cells such as the cell/cell contact, which is increased by immobilisation.
  • secondary metabolite syntheses since this system allows characteristic properties of differentiated tissue to be transferred.
  • the cells are no longer homogeneously mixed and grow as small calli or aggregates in connection with a differentiation of the cells.
  • the maintenance of stable, active biocatalysts is enabled by different degrees of the growth, since the risk of genetic instability of selected cell lines, and the loss productivity are reduced.
  • Non-limiting preferred examples of synthetic polymers are (poly)methylacrylate, (poly) styrene, (poly) acrylic ester, (poly) acrylamide, (poly) acrylonitrile, (poly) urethane, (poly)propylene, polypropylene oxide, nylon, silica, glass or ceramics .
  • Preferred examples of natural polymers are alginates, carrageenan, chitosan, chitin, pectin, agarose, agar, dextran, gelatine, cellulose sulphate and derivatives, proteins, such as for example poly-L-lysine or poly- - methionine as well as mixtures thereof.
  • the inclusion of plant cells in natural polysaccharides is the preferred method, since it is simple, cost effective and shows a reproducibly . Further, polysaccarides will preserve the cells .
  • the following table shows an overview of the preferred polymers for the immobilisation of plant cells and the gellation mechanism used.
  • Polyacrylamide Polymerisation polyacrylamide hydrazide Crosslinking polyphenylene oxide Crosslinking agarose-gelatine gel formation in the cold, crosslinking alginate-gelatine ionotropic gel formation, crosslinking alginate/chitosan ionotropic-polyelectrolytic coacervate formation ⁇ -carrageenan/chitosan ionotropic-polyelectrolytic coacervate formation pectin/chitosan ionotropic-polyelectrolytic coacervate formation
  • Cell immobilisation by adsorption is a further preferred method.
  • the cells are bound to a solid support.
  • any solid carrier can be used that enables the cells to immobilise.
  • These carriers comprise but are not limited to glass fibres, charcoal, nylon, resins, XAD, and amberlite.
  • the binding forces and the cell loads are relatively small. Therefore, this method is used for immobilisation of entire or parts of cells.
  • the advantage of this method is the free diffusion of nutrition agents to the cells and the fast removal of their products.
  • a further method of adsorption is the inclusion of the plant cells in honeycomb structures of films, ceramics or other supports. Plant cells can immerse spontaneously in the foam material, grow in the pores until they fill in the volume. This form of cell inclusion is the mildest and least detrimental method of all immobilisation techniques. Usually, the foams have a porosity of 90 %, high cell densities comparable to gel immobilisation can be achieved.
  • the cells of a suspension culture according to the invention usually do not grow as single cells, but form aggregates in the dimension of preferably 100-500 ⁇ m (few cells) up to some millimetres (up to thousands of cells) .
  • the formation of these larger cell aggregates results in a differentiation of cell structures.
  • the term "differentiation” means in this context a metabolic or morphological specialisation of cells.
  • the biochemical differentiation refers to the specialisation of metabolism and thereby depends on the coordinated expression of specific enzymatic biosynthetic pathways for the production of secondary metabolites.
  • Formation of aggregates in a production process may not always turn out to be advantageous, since the aggregates are susceptible to mechanic degradation.
  • a suspension culture is used for the purpose of the present invention.
  • a callus on a solid nutrition medium is the source material for a cell suspension culture.
  • Suspension cultures have considerable advantages compared to callus cultures. The culture is growing much faster and can be used like a culture of microorganisms. In suspension cultures, effectors (e.g. precursors and/or elicitors) can be applied more effectively compared to a solid nutrition medium.
  • cell aggregates are used in suspension cultures.
  • the term "cell aggregates” denotes aggregates comprising 20-100 cells, whereas fine suspension cultures comprise “aggregates” with 3-20 cells.
  • the nutrient medium is exchanged at least once during the production of the desired cyclic peptides, polypeptides or proteins and further in especially preferred embodiments it is exchanged at least once during the cultivating step.
  • the accumulation of secondary metabolites is usually dependent of three factors: Firstly, the product synthesis rate must be higher than the degradation rate, secondly the accumulated substances may not be toxic for the cells, and thirdly the products formed should not inhibit the continuous syntheses of the metabolites.
  • the product synthesis rate must be higher than the degradation rate
  • the accumulated substances may not be toxic for the cells
  • the products formed should not inhibit the continuous syntheses of the metabolites.
  • special accumulation cells or organs for secondary metabolites are lacking, so that the substances formed are usually accumulated intracellularly in the vacuoles and are toxic in higher concentrations for the cells or may lead to feedback inhibition. Therefore, the continuous release of accumulated products is necessary. Only a small amount of plant cell cultures can release the metabolites synthesised without external influences. An induced product release can be achieved by permeabilisation of plant cell membranes.
  • the cyclic peptides have to be removed and recovered from the culture after or during production of said cyclic peptides .
  • the recovery of said cyclic peptides, polypeptides or proteins from said cells or said medium of said cell or tissue culture, or both, is carried out sequentially or continuously.
  • the recovery of said cyclic peptides, polypeptides or proteins from the cells or the medium of the cell or tissue culture, or both, is carried out preferably by permeabilisation of the cell membranes of the cells of said cell or tissue culture.
  • Cell permeabilisation depends on the formation of pores in one or more membrane systems in the plant cell to enable the permeation.
  • the irreversible opening of biological membranes leads to a loss of the associationation of the cells, therefore to the release of toxic metabolites and lytic enzymes and therefore to the cell death.
  • the permeabilisation agents used within the scope of the present invention and the corresponding methods have to be used in such doses and/or concentrations, as to only cause a short time opening of the membranes which allows a closing by the natural movement (fluidity) of membrane lipids .
  • the permeabilisation methods can be classified in three groups, that is in chemical, physical and biological methods .
  • Preferred chemical agents including the release of metabolites are for example liquids like toluene, ether, dimethylsulfoxide, n-propanol, chloroform, phenethylethanol , ethanol, hexadecyltrimethylammonium- bromide, hexadecane, miglyol, antibiotics like nystatine, filipine, polyene antibiotica, polycations like poly-L- lysine, poly-L-ornithine, chitosan, proteins like cytochrome C, protamine sulfate, lipases, detergents like lysolecithine, Triton-X-100, Tween 20, saponines like digitonine, tomatine, calcium chelators as EDTA.
  • liquids like toluene, ether, dimethylsulfoxide, n-propanol, chloroform, phenethylethanol , ethanol, hexadec
  • Further methods using chemical agents include inducing osmotic pressure by adding mannitol, inorganic phosphates, etc. altering the ionic strength by a variation of an external pH value, addition of CaCl 2 , KC1 or K 2 S0 4 .
  • pores can be closed by the mobility of phospholipides in the membrane chains.
  • Molecular biological methods e.g. the integration of signal sequences in genes of metabolite production (for example peptides, proteins), which can be cleaved after a transport from the cell, enable the extracellular release of the desired peptides in the nutrition medium.
  • the recovered products include circulins, cycloviolins, kalatas, cyclopsychotrids, palicoureins, vitri, vodo, vico, varv peptids, hypa A cycloviolacin or McoTO.
  • Figure 1 shows the schematic representation of a device for performing a process for the production of cyclic peptides according to ' the invention.
  • Non axenic material of plants producing cyclic peptides was used. These plants belong to the families Rubiaceae, for example Oldenlandia affinis, Chassalia parvifolia, Psychotria longipes, Violaceae, for example Viola tricolor, Viola odorata, Viola arvensis, Leonia cymosa, Palicourea condensata and Curcubitaceae, for example Mormordica cochinchinensis .
  • Protoplasts were prepared from intact tissues (root, stem and leaf, fruits) but can also be prepared from callus and suspension cultures. Under suitable conditions, protoplasts can be cultivated over a long period. First they regenerate the cell wall and afterwards, the cell division occurs. Complete regeneration to a fertile plant is a prerequisite for use of protoplasts for plant cultivation.
  • the enzyme used for isolation usually cellulases and pectinases
  • the enzyme preparations are sometime toxic for some cell types. This can be avoided by desalting and subsequent lyophilisation . Contact of turgescent cells with the enzyme solution can eventually lead to cell death.
  • protoplasts Independent of the source, the isolation methods and the use, protoplasts have to be stored in highly concentrated solutions. Usually, sugars or sugar alcohols are used which are impermeable or nearly impermeable for the plasma lemma.
  • organs are used as protoplast source, it is necessary for the enzymes to address the cells in their entirety. In these cases, sometimes the lower epidermis can be removed. In the case of flower leaves for example, tissue is cut into fine particles in some millilitres of suitable plasmolyticums . For some uses it is required to decontaminate the organs before the isolation of the protoplast .
  • Protoplasts can be obtained from leaves of many species. It was shown, that the physiological state of the plant is extremely important for quality, yield and regeneration possibility of protoplasts.
  • Preferred conditions are:
  • a low light intensity 500 to 1000 ⁇ W/m 2 ) , change of light and dark, temperature of 20-25 °C, relative humidity of 60- 80 %.
  • Calcoflour white ST specifically binds to ⁇ -1, 4-glucan . In UV light at 430 nm, the product fluoresces brightly and thus allows the determination of cell wall material. Calcofluor White ST, Sigma or Fuchsin methyl blue, Sigma was used to stain remained cell wall of protoplasts.
  • the viability of plant cells and protoplasts was determined either by physiological indicators or by membrane semipermeability characteristics of vitality.
  • the production described herein is a modified version of Horsch et al . , 1985.
  • the transformation is carried out by the leaf-disc method. 10 ml of a culture of a A. rhizogenes single colony (wild type) are pelletised at 4000 g and resuspended in 20 ml of Yeb's liquid medium. Leafs of germ free cultivated plants of the line to be transformed are cut in 1-2 cm 2 pieces and immersed for 2-3 minutes in the agrobacteria suspension. The leaf pieces were placed on solid medium and co-cultivated for two days in the dark at 24 °C with the agrobacteria.
  • the co-culture phase enables the insertion of the agrobacteria in the tissue and the stable integration of the alien DNA in the plant genome. Subsequently, the pieces of leafs were placed on a selective medium in cultivated with a light-dark-rhythm of 16 hours to 8 hours. The hormones in a medium induce the formation of the callus tissue where hairy roots can be regenerated.
  • the agrobacterium By replacement of a tumour causing gene of T-DNA by extrinsic genes, the agrobacterium cannot induce further formation of tumours and is used exclusively as a gene transporter and enables the integration of alien genes or additional strong plant promotors and operated genes in the plant genome.
  • the reporter gene In the T-DNA vectors used for the plants transformation, the reporter gene is present in one, two or three copies .
  • organogenesis means the formation of shoots or of roots from callus or cell cultures.
  • the regeneration to intact plants can be achieved in different ways: 1. organogenesis, 2. somatic embryogenesis .
  • organogenesis With callus or already differentiated material, it can regulated by the use of phytohormones if more stems or roots are formed or if non differentiated callus is formed.
  • a screening program essentially known to an artisan can be used to find the optimum composition of the nutrition medium with respect to the phytohormones.
  • BAP 6- benzyle aminopurine
  • NAA 1-naphthyl- acetic acid
  • Both are synthetic products.
  • different materials can be used for example freshly isolated callus, shoot cultures, explants, the organogenesis of callus works best with freshly developed cultures (not over the half year) .
  • a decisive factor for the formation of roots and shoots with stem explants is the composition of the nutrition media with respect to a phytohormone .
  • An excess of cytokinines stimulates the formation of leafs and sprouts.
  • a sterile shoot culture, which is grown by adding an excess of cytokinines can be rooted with auxines. Thereby, entire plants are obtained which can be planted in earth.
  • the basic technique of tissue and cell culture research is the establishment and further cultivating of a callus culture.
  • Decontaminated parts of the tissue (explants) were grown on cultures, which increase the formation of callus.
  • the culture comprises phytohormones (for example auxine (NAA) and cytokinine (BAP)) in suitable concentrations, undifferentiated cell growth occurs in several parts of the explants.
  • a callus will arise from such non differentiated cells. Differentiated and necrotic parts are excised, and the callus can be cultivated over years independent from the donor plant.
  • This callus is also the basic material for suspension or cell culture.
  • the cells are totipotent. Changing the composition of the phytohormones, the concentration of e.g. sucrose and eventually also the light might lead to a redifferentiation of the callus cells.
  • the decontamination of plant organs was achieved by a treatment with solutions of hypochloric acid. It is preferred to use a short pretreatment with ethanol to increase the wettability of the epidermis (cuticula) or an edition of detergence. Irradiation
  • the irradition with irradiation sources of different wave lengths and energy is a very important factor for carrying out the present invention. Further important factors in inducing expression in Viola species is the composition of the medium, the influence of light and the addition of phytohormones. If the medium is a MS phase, vitamins (biotin) 50 ⁇ g/1, and folic acid (500 ⁇ m/1) were added. Furthermore, hormones (2,4 D, 0,4 mg/1 BAP and 3 mg/1) were added. The light source was a Osram Lumilux cool white with an intensity of 30 ⁇ Em ⁇ S "1 .
  • the corresponding value for the induction of expression with Oldenlandia species carried out in a medium (MS phases) by addition of hormones (BAP 2,25 mg/1 and NAA 0,186-1,86 mg/1).
  • the irradiation source was a Sylvania Grolux (F15/GROT8) with an intensity of 5 to 9 ⁇ Eirf's "1 .
  • hormones (NAA 1 mg/1) were added and the irradiation source was an Osram Flora 15 with an energy of 25 ⁇ Em ⁇ S '1 .
  • the callus cell was separated with the spatula from the explant and placed on fresh medium. Incubation occurs in Petri dishes closed with parafilm at room temperature in the dark.
  • the plant parts were cleaned and washed with tap water. Fresh cut pieces (ca. 50 mm long) were rinsed for 1 minute with 70 % ethanol.
  • the pieces were taken under sterile conditions and transferred into diluted sodium hypochlorite solution. After decontamination times of 30, 60 and 90 minutes they were washed three times in sterile deionised water.
  • the roots, stems, leaves, etc. were cut in about 0.5 cm thick portions and placed on different callus induction media (MS, B5 (Gamborg et al . , 1968)).
  • the flasks were closed with a parafilm and incubated at room temperature in the dark and under light.
  • the stems of the plants were cut into pieces of 30-50 mm and put in the medium.
  • M-medium based on MS with sucrose concentrations of 0 - 20 g/1 without additional phytohormones. Cultivation was carried out at 24 °C and 40 ⁇ E/m 2 . After two weeks, a new shoot is formed and roots were also formed.
  • the mannite solution was removed from the flask and 12 ml of 0.5 M mannite solution for a preplasmolysis of 1-2 hours at room temperature was added.
  • the mannite solution was removed and was replaced by 12 ml enzyme solution (mazerocyme R-10, 0.6 units/mg: 0.25 % and cellulase "Onozuka R-10", 1.55 units/mg: 1 %, 8 mM CaC12 0.4 M mannite, pH 5.5, sterile filtrated) .
  • Incubation was carried out during 18-22 hours at 25 °C in the dark.
  • the protoplast suspension produced by the cell was removed and filtrated through two sieves (sieve hole diameters: 125 ⁇ and 63 ⁇ m) .
  • the flask was rinsed with 6 ml of 0.2 M CaCl2 solution which was given to the filtrate through the sieves.
  • the suspension was separated in two and placed in two centrifuge tubes and was centrifuged for 5 minutes and 600 rpm (235xg) . After removal of the supernatant, the pellet was resuspended with 3 ml 0.5 M mannite solution and 6 ml 0.2 M CaC12 solution and centrifuged at 600 rpm. The pellet was resuspended with 6 ml of 0.5 M mannite solution and 3 ml 0.2 M CaCl 2 solution and once again centrifuged. The last pellet was resuspended with 5-10 ml in W5 solution (145 mM NaCl, 125 mM CaCl 2 , 5 mM KC1, 5 mM glucose, pH 5.6-6.0).
  • the conditions for isolation have to be optimised for each plant.
  • a person skilled in the art is fully aware that besides the specifically mentioned conditions that have to be observed other conditions can be used without deporting form the scope of the present invention. This can be determined in a few routine experiments without undue burden by respecting the following parameters: low enzyme concentration, temperature approx 20 °C, correlated with long incubation times (long time isolation) and high enzyme concentration, temperature around 25-30 °C, mixing, correlate to short incubation times (short time isolation).
  • pectinase and cellulase can also be used subsequently (two step method).
  • the quality and purity of the protoplasts are determined by the examination of microscopic images taken therefrom.
  • the concentration of the content of the cyclic peptides can be determined after dry refrigeration of the biomass and extraction (solid-liquid) with methanol under stirring at room temperature.
  • the methanolic extract was further extracted with hexane (the hexane extract was discharged) followed by a liquid- liquid extraction of the methanolic extracts with butanol .
  • the butanol phase comprised the cyclic peptides and was dried.
  • Cultures were taken from the cultivation flask, cut in a flask and the leaves are cut off. From longer internodia, 5-8 mm long pieces are cut and placed with the physiological face to the periphery of the flask on the nutrition medium and put in the agar. In each flask, three to five stem pieces were placed. The flasks closed with parafilm. Cultivation was carried out at 24 °C and 40 ⁇ E/m 2 .
  • the cells with a differentiation were be stabilised by the selection of modified medium in suspension/liquid media.
  • Concentrations of auxine and cytokinine combinations were usually in the range between 0-10 '10 M preferably 10 "7 to 10 "5 M.
  • 150 ml of a cell suspension were placed in a flask with 550 ml of medium and medium with chitosan (for example 0, 50, 100, 250 ⁇ g/ ml) .
  • chitosan for example 0, 50, 100, 250 ⁇ g/ ml
  • the elicitor was added after a culture time of 4-7 days. After 7-21 days, the cell cultures were harvested and examined. The cell suspensions were separated under vacuum by a B ⁇ chner filter from the medium. Cells and medium were investigated separately.
  • the product extraction was carried out after lyophilisation of the biomass and extraction (solid-liquid) with methanol under stirring at room temperature or by liquid-liquid extraction of the medium. The methanolic extracts were extracted with hexanes (the hexane extract was discarded) and subsequently the liquid-liquid extraction of the methanolic extracts were carried out with butanol.
  • the butanol phase contains the desired cyclic peptides and was dried.
  • the immobilisation is achieved by coacervate formation with pectin and chitosan:
  • 25 g of 5 % pectinic acid (1.25 g pectin/25 g medium) are mixed with 5 g Oldenlandia cells.
  • the polysaccharide cell mixture is added under sterile conditions drop by drop to 80 ml of a chitosan hardening solution (1 % chitosan, 0.8 % CaCl 2 in deionised water) and incubated during 30 minutes under stirring.
  • a chitosan hardening solution (1 % chitosan, 0.8 % CaCl 2 in deionised water
  • the immobilised cell cultures were washed afterwards for three times with a sterile medium.
  • the immobilised cell cultures are used as biocatalysts for the production of cyclic peptides preferably in a continuous perfusion system.
  • a plant cell culture for example a non-differentiated suspension culture or immobilised cells of Viola tricolor
  • the process was in the production phase of the target peptide.
  • the induction of product expression was introduced by addition of elicitors or precursors.
  • the production of the target peptide is induced thereby.
  • the peptides will then be secreted or purified after application of specific permeabilisation strategies.
  • Microfiltration coupled to the bioreactor allows separating the peptide continuously or sequentially. Further microfiltration allows for the concentration of the product solution and the separation from low molecular media compounds.
  • the ultimate purification was carried out by chromatographic methods by use of a FPLC system.
  • Device 100 comprises a plant cell culture bioreactor 101 which is for example described in DE 197 47 994 Cl or in EP 0 911 386 A2.
  • bioreactors 101 suitable for use in the present invention are for example air-lift reactors packed bed reactors, where beads, capsules or adsorption resin or aggregates of embryonic cultures are used, fluid bed reactors, stirred tanks, conventional reactors for heterotrophic cell cultures, photobioreactors for photoautotrophic and photoorganotrophic (mixotrophic) cell cultures etc.
  • Bioreactors 101 can be operated in continuous or discontinuous operation modes, dependent on cell growth and peptide production. If immobilised cell cultures are used, replacement rates depend only on the production of the peptides.
  • the irradiation source 102 or a combination of one or more irradiation sources can either be an integral part of the bioreactor 101 or can be an isolated part which is connected to the bioreactor 101.
  • every light source providing the desired wavelengths and energy density can be used.
  • the preferred energy (expressed as energy density) of the irradiation source 102 is preferably in the range of 5 - 30 ⁇ Em ⁇ 1 .
  • Preferred light sources have specific wavelength spectra. It is important that the spectrum comprises wavelengths in the range of photosynthetic light like 650 - 700 nm.
  • the irradiation protocol is usually 16 h irradiation followed by 8 h darkness as often as required.
  • Light having the aforementioned energy density and/or wavelength has a decisive influence on the cell growth and biochemical differentiation (formation of Chlorophyll) which is very important for the production of cyclotides. Without light, or light of different wavelength and/or energy density, lesser amounts of cyclotides are formed.
  • UV-A 315 - 380 nm
  • UV-B 280 - 315 nm
  • specific examples of commercial available light sources are for example: Ultravitalux, Eversum, Ultramed all by Osram) light which have regulatory effects on the formation of different metabolic products in the cell (amino acids similar to mycosporin) and cell growth.
  • Red-light near 655 - 665 nm, for 725 - 735 nm
  • the plant cell substrate is prepared and stored in a vessel 103 and is transferred via pump 104 to bioreactor 101.
  • the plant cell or tissue culture is preferably derived from Violaceae, Cucurbitaceae species and cultivated as described before.
  • the bioreactor 101 is further equipped with a stirrer 105 which is in some embodiments a mechanical stirrer, whereas any other stirrer can be used which is capable of continuously and homogeneously stirring the suspension.
  • Compressor 119 generates compressed air which is introduced in the bioreactor 101 via valves 120 for a better mixing.
  • the mechanical stirrer 105 is omitted and mixing occurs only via compressed air.
  • the peptide-containing suspension is transferred to a vessel containing the permeate 108 and is afterwards mixed in the mixing vessel 109 with butanol or any other suitable solvent, which is stored in vessel 110 and added via pump 111 to the mixing vessel 109.
  • the mixture is transferred to a settler 112 and the phases are separated.
  • the aqueous phase is transferred via pump 113 to waste vessel 114 where the aqueous phase is discarded.
  • the butanolic phase is transferred via pump 115 to a vessel 116 where the butanolic phases containing the cyclic peptides are collected.
  • the collected butanolic phases containing the cyclic peptides is transferred via valve 117 over a fast protein liquid chromatographic column 118 and the purified cyclic peptides are recovered for further downstream and use.

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Abstract

La présente invention concerne une méthode destinée à la production de peptides, de polypeptides ou de protéines cycliques dans un système de culture in vitro d'espèces d'origine végétale de la famille des violacées, des cucurbitacées ou des rubiacées. Cette méthode consiste (1) à cultiver des cellules ou des tissus végétaux dérivés de cultures de cellules isolées ou de suspensions de cals, d'organes, d'embryons, dans un ou plusieurs milieux nutritifs, (2) à induire l'expression d'un ou plusieurs desdits peptides, polypeptides ou protéines cycliques et (3) à récupérer un ou plusieurs desdits peptides, polypeptides ou protéines cycliques à partir desdites cultures de cellules ou de tissus ou dudit milieu de culture de cellules ou de tissus, ou des deux, lesdits peptides, polypeptides ou protéines cycliques comportant un noeud de cystéine cyclique ou son équivalent chimique ou structural et au moins trois liaisons disulfure.
EP05739387A 2004-05-07 2005-05-09 Methode de production de peptides cycliques a partir de cultures cellulaires vegetales in vitro Withdrawn EP1747278A1 (fr)

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PCT/EP2005/004999 WO2005108596A1 (fr) 2004-05-07 2005-05-09 Methode de production de peptides cycliques a partir de cultures cellulaires vegetales in vitro

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US7211658B2 (en) 2004-10-05 2007-05-01 E.I. Dupont Denemours And Company Insecticidal plant cyclotide with activity against homopteran insects
MY157759A (en) * 2006-11-28 2016-07-15 Univ Namur Composition comprising oligogalacturonans and polycationic saccharides
CH715456B1 (de) 2007-04-27 2020-04-30 Mibelle Ag Kosmetisches Produkt zur topischen Anwendung für den Schutz und die Erneuerung von Hautstammzellen, welches sich von dedifferenzierten Pflanzenzellen ableitet.
EP2687592A1 (fr) 2012-07-20 2014-01-22 greenovation Biotech GmbH Filtration de surnageants de cultures cellulaires
EP3808335B1 (fr) 2019-10-15 2024-05-22 Faes Farma, S.A. Compositions pour administration topique
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same
CN117730762B (zh) * 2024-02-19 2024-05-03 浙江省生态环境科学设计研究院 一种促进絮凝剂胁迫下沉水植物生长的方法

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Title
See also references of WO2005108596A1 *
SEYDEL ET AL: "Gewinnung von Viola tricolor-Zellkulturen zur Produktion zyklischer Peptide", DECHEMA WORKSHOP, PHYTOEXTRAKTE - PRODUKTE UND PROZESSE (POSTER), 2004, FRANKFURT *

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