EP1747278A1 - Method for producing cyclic peptides from in vitro plant cell cultures - Google Patents

Abstract

The present invention relates to a method for producing cyclic peptides, polypeptides or proteins in an in vitro culture system of plant origin Violaceae species, Cucurbitaceae species or Rubiaceae species comprising the following steps: (1) cultivating a plant cell or tissue culture derived from callus, organ, embryonic, suspension or single cell cultures, in one or more nutrient media, (2) inducing expression of one or more said cyclic peptides, polypeptides or proteins, (3) recovering one or more said cyclic peptides, polypeptides or proteins from said cell or tissue culture or said medium of said cell or tissue cultures, or both and wherein the cyclic peptides, polypeptides or proteins have a cyclic cysteine knot or its chemical or structural equivalent and at least three disulfide bonds.

Description

Method For Producing Cyclic Peptides From In Vitro Plant Cell Cultures

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.

The use of plant cell cultures is known for several decades. Further, the cultivation of suspension cultures or the in vitro regeneration of plants is also known for a long time. 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) .

Higher plants provide a large spectrum of secondary metabolites for example peptides with clinical and pharmaceutically interesting properties and they further provide specific flavour colour or antioxidative properties or can be useful as biocides (Fowler, M.W. & Scragg, A.H. in: Plant Cell Biotechnology, Springer Verlag, Berlin, Heidelberg, 1988, page 165 - 177) .

Many of these substances cannot be synthesised by microbiological, enzymatic or chemical syntheses because their chemical structures are too complex. Usually, the plant material for obtaining these products is cultivated or collected. The in vitro production of important natural products by the use of plant cultures and recombinant cells provides in analogy to a microbial production the advantage of being independent from biotic and abiotic factors and is therefore accessible to standardisation.

The industrial use of cell and tissue cultures for the production plant compounds requires an optimisation and an efficient increase of product yields. For example, this requirement is met by the selection of highly productive cell lines, by variations of media and cultivation regimes or, alternatively, by the use of specific strategies for improving the yields of plant cell cultures.

The ability of plant cell cultures to divide, grow and produce the secondary metabolites under a variety of different cultural regimes has been amply demonstrated by a number of research groups. Strategies for resolving the problems in non differentiated plant cells comprise the optimisation of media, the induction or stimulation of biosynthesis by elicitors or precursors and the optimisation of culture conditions (for example, by specifically optimizing factors like light, temperature, etc . ) .

Especially for a large scale' production, 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.

Technological fermentation processes are typically carried out, by discontinuous or continuous cultures and variations of the downstream processing. Stafford et al . (1986) provide a detailed overview on the influence of the optimisation of media and the importance of physical parameters. Due to the high specification of plant cell cultures, cultivation conditions avoiding shear forces are necessary. The cultivation of many different plant cultures in stirred tanks is only practised with limited success due to the resulting shear forces.

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 Dδrnenburg and Knorr, Proc. Biochem. 27 (3 ), 161-166, 1992 or Dδrnenburg and Knorr, Enzyme Microb. Technol . 17(8), 1995, 674-684.

The use of three phase systems for in situ product removal is also known (in situ product adsorption or product extraction) .

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. In many proteins, the linear peptide backbone is cross-linked via disulfide bonds between cysteine residues. Usually, 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. Notable examples of these small 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) .

Methods for the production of such cyclic peptides have been so far not be scaleable on an industrial level.

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.

This problem is solved by a method for producing cyclic peptides, polypeptides or proteins in an in vitro culture system of plant origin comprising the following steps:

(1) cultivating a plant cell or tissue culture derived from callus, organ, embryonic, suspension or single cell cultures derived from a Violaceae species, Curcurbitaceae species or Rubiaceae species, in one or more nutrient media (2) inducing expression of one or more said cyclic peptides, polypeptides or proteins by applying endogenous or exogenous cell stress

(3) recovering one or more said cyclic peptides, polypeptides or proteins from said cell or tissue culture or said medium of said cell or tissue cultures, or both and wherein the cyclic peptides, polypeptides or proteins have a cyclic cysteine knot or its chemical or structural equivalent and at least three disulfide bonds. Preferably, 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 .

In a further preferred embodiment, said cyclic peptides, polypeptides or proteins comprise a beta-hairpin structure, other preferred structures are moebius and non-moebius structure .

It is especially preferred that 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. In preferred embodiments, the energy of the light source is in the range of 10 to 100 μEm^"2S^_1, more preferred from 20 to 15 μEm^S^-1, and most preferred from 25 to 35 μEm^"2S^_1. In the case of Violaceae species this value is in the range of 30 μEirf²S^-1. It is understood, that also 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 .

In a further preferred embodiment, 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

In further preferred embodiments 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.

It is preferred to use 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. There are many examples in the literature demonstrating a relationship between differentiation and secondary metabolic accumulation.

Differentiated cell cultures are characterised by the ability of cell division and regeneration of new tissue.

Typically 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. For example, 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). Sometimes, 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.

The term "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.

Important factors in adding elicitors are the concentrations of the elicitors and the protocol of adding the elicitors. In different embodiments of the present invention, 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 μg/ml. Jasmonic derivatives are added in a range from 10 to 200 μg/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.

If 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 HgCl₂, CuS0₄, 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.

Mechanical injury causes the removal of pectin fragments from the plant cells. Such removal is considered in the context of the present invention as an endogenous elicitor.

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, β-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 β-1 , 4-N-acetyl-D-glucosamine and has variable positive charges depending on the degree of acetylation.

It is important to note, that as explained before also the concentration of such elicitors plays an important role. Non-limiting examples for polysaccharide elicitors and their respective preferred concentrations are given in table 1.

Table 1: Polysaccharide-Elicitors Polysaccharide Concentration [μg/mL]

Agar 500-20000

Agaropectin 10-500

Agarose 2000-20000

Alginate 500-1000

Ca-Alginate 10000

Na-Alginate 10000

Chitosan 150

Chitosan 0,2-25

Chitosan 500

Curdlan 200

Gellan 2000-20000

Oligogalacturonic acid 500

Pectic acid 5-500

Xanthan 200

Curdlan/Xanthan 100/100

Addition of appropriate precursors or related compounds to the culture media further stimulates secondary metabolite production. This approach is especially advantageous if the precursors are inexpensive. Examples of 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. In this context, 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 .

In some preferred embodiments of the present invention, precursors are also added during the addition of elicitors to support cells in the production of the cyclic peptides. In another variant of the present invention, precursors are added from the very beginning in the medium in continuous protocol, since for example the reduced precursors are sometimes not stable.

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.

In a further preferred embodiment of the invention, 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.

Preferably, the immobilisation supports a continuous process; the biocatalyst can be used more often and is easier to separate from the cultivation medium. Apart from these advantages, there are further ones specifically useful for plant cells, such as the cell/cell contact, which is increased by immobilisation. There are advantages for the 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. Therefore, it is possible to stabilise the metabolism of plant cells through immobilisation by transferring them in a silenced state. In many cases, by immobilisation alone, the production and secretion of normally stored metabolites can be induced. Further, plant cells are protected by the immobilisation as support for rheological forces. Most preferred is an immobilisation by embedding the cell or tissue culture in natural or synthetic polymers.

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.

Table 2 : Preferred polymers for the immobilisation of plant cells

POLYMER GELATION MECHANISM

Agar temperature-induced gelling

Agarose ionotropic gel formation

Alginate ionotropic gel formation κ-carrageenan ionotropic gel formation

Chitosan ionotropic gel formation

Gelatine gel formation in the cold, crosslinking

Gellan ionotropic gel formation, gel formation in the cold

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 κ-carrageenan/chitosan ionotropic-polyelectrolytic coacervate formation pectin/chitosan ionotropic-polyelectrolytic coacervate formation

Cell immobilisation by adsorption, is a further preferred method. Through means of adsorption, the cells are bound to a solid support. Within the scope of the present invention, 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.

It was found that immobilisation has a considerable impact on the physiology of the cell and improves the biosynthetic yield of immobilised cell cultures.

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 μm (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.

These large aggregates can be regarded as naturally immobilised cell systems which do not use an artificial matrix (for similar concepts see for example Fuller and Bartlett, Ann. Proc . Phytochem. Sub. Eur, Vol. 26, 1985, 229-247) .

Formation of aggregates in a production process may not always turn out to be advantageous, since the aggregates are susceptible to mechanic degradation.

In further preferred embodiments 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.

In an especially preferred embodiment, 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. In the process according to the invention, 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. In non differentiated plant cell cultures, 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 . In preferred embodiments 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 compartimentation of the cells, therefore to the release of toxic metabolites and lytic enzymes and therefore to the cell death. For this reason, 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 .

If chemical agents are used, the maintenance of cell vitality is a problem. The permeabilisation step, which is used for a certain period for the release of the product requires a subsequent washing step of the cells for the removal of toxic agents. Permanent treatments with chemical substances in small ^' concentrations are not known to date. 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. 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 CaCl₂, KC1 or K₂S0₄.

Physical techniques for permeabilisation (freeze-thaw cycles, electroporation, temperature, high hydrostatic pressure, ultrasonic treatment, iontophoresis) have the advantage, that at the end of the treatment, no further effect on the cell cultures is caused, and the cells can regenerate themselves without external influence. These methods can be used for a cyclic process for the intermitting product release. In a further preferred embodiment electroporation is used which is a method for destabilisation of cell membranes, wherein the cells are exposed to electric pulses and therefore, specific regions in the cell membranes can be destabilised.

Further preferred is the application of pressure where membranes show usually a change in permeability. Malfunction in plant membranes induced by pressure is for example an inhibition of amino acids transport due to denaturation of membrane proteins .

The use of biological methods allows compared to the chemical and physical permeabilisation techniques a targeted approach to influencing the plant membrane system and thereby to the targeted opening of the membranes . In the present context, 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.

Further advantages and preferred embodiments will become evident from the following description of the examples and the figure.

It is understood, that the examples are intended for illustrative purposes only and are not meant to be limiting for the scope of the present invention.

Figure 1 shows the schematic representation of a device for performing a process for the production of cyclic peptides according to^' the invention.

Experimental

General

For the production of in vitro plant cultures, 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 .

It is understood that instead of the flasks described in the following other appropriates means like bioreactors, Petri dishes etc. can be used. Examples of suitable media are MS (Murashige + Skoog) and B5 media (Gamborg et al . ) which are well known to an artisan. It is understood that any other medium suitable for the purpose of the present invention and known to an artisan can be used as well.

Protoplasts for generation of callus cultures and in vitro plants

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) have a great impact on the yield quality of the protoplast. Different methods for the isolation are required, such as enzyme mixtures and concentration, temperature, time of incubation etc.. 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. Therefore a plasmolysis is preferred. 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.

If intact 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 μW/m² ) , change of light and dark, temperature of 20-25 °C, relative humidity of 60- 80 %.

Cell and Viability Tests

The efficiency of cell wall degradation and the viability of the cells and developed protoplasts were determined by simple staining tests.

Calcoflour white ST specifically binds to β-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 Monitoring of Protoplast Viability was carried out with Evan's Blue and Neutral Red using the following procedure:

After addition of a drop of neutral red (0.1% in 0.7 M sorbitol) to a drop of the protoplast suspension, neutral red readily passes through membranes and accumulates in the vacuole. Living protoplasts appeared with red colour caused by the accumulation of neutral red.

After addition of a drop of Evan's blue (0.5% in 0.7 M sorbitol) to another drop of protoplasts (on a regular slide) , viable protoplasts will appear white or yellowish against a blue background, no accumulation occurs.

Production of "Hairy Root" cultures

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 cm² 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.

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. In the T-DNA vectors used for the plants transformation, the reporter gene is present in one, two or three copies .

The term "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 . 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. For different plants species, 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. In the following, the influence of 6- benzyle aminopurine (BAP) as cytokinine and 1-naphthyl- acetic acid (NAA) as auxine and a combination of both phytohormones is described. Both are synthetic products. As starting materials, 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) .

Plant cultures

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 . Usually the ratio of cytokinines and auxines is a decisive factor. 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. For obtaining genetically stabilised material, it is desirable, that the organs are formed without the interface of a callus.

Callus cultures (non differentiated cell culture)

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. If the culture comprises phytohormones (for example auxine (NAA) and cytokinine (BAP)) in suitable concentrations, undifferentiated cell growth occurs in several parts of the explants. By subsequent separations, 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 μg/1, and folic acid (500 μm/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 μEm^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 μEirf's^"1.

Upon using a B5 basis medium, hormones (NAA 1 mg/1) were added and the irradiation source was an Osram Flora 15 with an energy of 25 μEm^S^'1.

1. Surface decontamination of Viola tricolor seeds

100 seeds were rinsed over 5 minutes in 70 % ethanol. The seeds were taken under sterile conditions and transferred in diluted in sodium hypochlorite solution. After decontamination times of 15, 30, 60 and 120 minutes, each 25 of the seeds for each time were transferred in sterile deionised water and washed three times. The decontaminated seeds are then placed with a sterilised tweezer in two flasks with water agar (8 g/1 agar) and MS-medium (Murashige and Skoog, 1962) without phytohormones (5-8 seed per platelet) and incubated at room temperature in the dark. The production of plant explantates for the formation of callus is made under sterile conditions. Plant sprouts are cut in 0,5-1 cm long pieces and put on medias (callus induction media with different nutrient and phytohormone combinations) . Flasks are closed with parafilm and incubated in the dark at room temperature.

After 7-14 days, a primary callus formation was observed. For subcultivation of the callus culture, 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.

2. Surface decontamination of non axenic plant parts of Leonia cymosa

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.

3. Propagation of a shoot culture of Oldenlandia affinis as donor plant for cell culture

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 μE/m². After two weeks, a new shoot is formed and roots were also formed.

4. Induction of callus and suspensional cultures from germ free shoot cultures A germ free plant was cut in 0.5 long pieces and put on callus induction media (for example B5) or in liquid media preferably 25 ml in a 100 ml flask. Incubation was carried out in the dark at room temperature (24 °C) in the liquid nutrition media at 100 rpm.

5. Isolation of protoplasts from Viola plants

1 g of young leafs (approx. 10 leafs) of germ free grown plants were cut in fine pieces in 0.5 M mannite solution in a Petri dish.

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.

After the preplasmolysis, 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 μ and 63 μm) . 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 CaCl₂ solution and once again centrifuged. The last pellet was resuspended with 5-10 ml in W5 solution (145 mM NaCl, 125 mM CaCl₂, 5 mM KC1, 5 mM glucose, pH 5.6-6.0). Usually, 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).

Alternatively, instead of the aforementioned one step method, 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.

6. Suspension cultures of Oldenlandia affinis and Viola tricolor

Cells of the cultures were removed from the respective flasks. 3 ml each of a thick cell suspension were placed in flasks with 25 ml of fresh nutrition solution and incubated at 24 °C and 100 rpm over a period of three weeks. The inoculum is up to 10 % ; for 1 1 cultures, 100 ml are used. Preferably 500 ml flasks are used with 100 - 120 ml of medium. The incubation time for the preculture was between 1 to 3 weeks . The initial suspensions were characterised by their pH value, connectivity, fresh cell weight, dry weight, and the concentrations of cyclic peptides.

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.

7. Organogenesis with Chassalia parvifolia stems

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 μE/m².

After 3-6 weeks, 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.

8. Increase of an axenic shoot culture of Viola arvensis

On the bottom of a sterile flask, shoots are cut in pieces of 30-50 mm length (with nodia) and these are put with a tweezers with the basal end in the nutrition medium. M medium (based on MS) with sucrose concentrations of 0-20 g/1 without the addition of phytohormones were used as the medium. The cultivation is carried out at 24 °C and ca . 40 μE/m². After two weeks new stems and roots were formed. For Viola plants, gellan instead of agar was used for solidification of the nutrition medium.

9. Increase of product syntheses in Oldenlandia affinis suspension cultures using elicitors

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 μg/ ml) .

Alternatively, 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 Bϋchner 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.

10. Production of cyclic peptides in plant cells after cysteine addition 0.1 % cysteine for example in form of the water soluble acetyl cysteine were added to the medium.

After 2-21 days of incubations, cells and medium are investigated with respect to cyclic peptides with disulfide bridges .

11. Immobilisation of Oldenlandia affinis suspension cultures in capsules

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 % CaCl₂ in deionised water) and incubated during 30 minutes under stirring.

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.

12. Production of cyclic peptides in an integrated bioprocess

After generation of 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.

A preferred device for carrying out the process of the invention is described in the following. It is understood, that any other suitable device with the essential elements as defined below is also suitable for the purposes for carrying out the process of the present invention.

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. Examples for further 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.

For the purposes of the present invention it is crucial, that located in the vicinity of the bioreactor 101 one or more irradiation sources 102 are present. 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. In principle, 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 μEm^δ¹. 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.

Further important parts of the spectrum are wavelengths in the range of UV-A (315 - 380 nm) and 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) (Grolux by Osram) has regulatory effects on the formation of different metabolic products in the cell, for example anthocyanin. It is understood that one or more of the aforementioned light sources can be used alone or in combinations thereof . 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. In some embodiments the mechanical stirrer 105 is omitted and mixing occurs only via compressed air. After having cultivated the plant cell or tissue culture an induced expression either by adding elicitors and/or precursors or irradiating or changing the nutrient medium, the peptide-containing suspension is removed from the bioreactor 101 via pump 106 to a micro- filtration device 107. After microfiltration in the microfiltration device 107, 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. After mixing the permeate with butanol, 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.

Claims

PATENT CLAIMS

1. A method for producing cyclic peptides, polypeptides or proteins in an in vitro culture system of plant origin Violaceae species, Cucurbitaceae species or Rubiaceae species comprising the following steps:

(1) cultivating a plant cell or tissue culture derived from callus, organ, embryonic, suspension or single cell cultures, in one or more nutrient media (2) inducing expression of one or more said cyclic peptides, polypeptides or proteins by applying exogenous or endogenous cell stress (3) recovering one or more said cyclic peptides, polypeptides or proteins from said cell or tissue culture or said medium of said cell or tissue cultures, or both, wherein and the cyclic peptides, polypeptides or proteins have a cyclic cysteine knot or its chemical or structural equivalent and at least three disulfide bonds.

2. The method according to claim 1, wherein the plant cell or tissue culture is derived from Chassalia species, Oldenlandia species, Psychotria species, Palicourea species, Viola species, Leonia species, Hybanthus species, or Momordica species.

3. The method according to claim 1 or 2 , wherein the plant cell or tissue culture is recombinant or non recombinant.

4. The method according to claim 3, wherein the plant cell culture is a differentiated or an undifferentiated cell culture system.

5. The method according to any one of the preceding claims, wherein the plant cell culture is a suspension culture, hairy root culture or embryonic culture.

6. The method according to claim 5, wherein the plant cell or tissue culture is cultivated by a batch, fed-batch, repeated fed batch or in a continuous perfusion modus process .

7. The method according to any one of the preceding claims, wherein the induction of expression of said peptides, polypeptides or proteins in step 2) is carried out by changing the composition of the nutrient medium.

8. The method according to any one of claims 1 to 6, wherein the induction of the expression of one or more of said cyclic peptides, polypeptides or proteins in step 2) is carried out by adding an elicitor and/or a precursor to the nutrient medium.

9. The method according to claim 8, wherein the elicitor is selected from the group of biotic elicitors comprising 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, β-cyclodextrin, locust beam gum, alginate and from microorganisms: alginate, rhamsan, xanthan, chitin, chitosan, curdlan, levan, gellan, welan.

10. The method according to claim 8, wherein the elicitor is selected from the group comprising metal compounds like HgCl , CuS0₄, inhibitors of protein syntheses like actinomycine D or cycloheximide, respiratory inhibitors like CN^~ or 2 , 4-dinitrophenol , pesticides and detergents, ozone, herbicides, e.g. acifluorfen, alpha-amino butyric acid, nitric oxide, sodium nitroprusside, silicium oxide.

11. The method according to claim 8, wherein the precursor is a primary metabolite.

12. The method according to claim 11, wherein the precursor is a sulphur containing amino acid, preferably cysteine or cystine or reductive compounds like glutathione.

13. The method according to any one of the preceding claims, wherein the plant cell or tissue culture is immobilised.

14. The method according to claim 13, wherein the immobilisation is carried out by embedding said cell or tissue culture in natural or synthetic polymers.

15. The method according to claim 14, wherein the natural polymer is a polysaccharide.

16. The method according to claim 13, wherein the immobilisation is carried out by adsorption of the cell or tissue culture on a solid carrier.

17. The method according to any one of the preceding claims, wherein the nutrient medium is exchanged at least once during the expression in step 2 of said cyclic peptides, polypeptides or proteins.

18. The method according to any one of the preceding claims, further comprising the step of exchanging the nutrient medium at least once during the cultivating step 1) •

19. The method according to any one of the preceding claims, further comprising the step of recovering of said cyclic peptides from the culture during the expression of said cyclic peptides.

20. The method according to any one of the preceding claims, wherein the step of recovering of said cyclic peptides, polypeptides or proteins from said cells or said medium of said cell or tissue culture, or both, occurs sequentially or continuously.

21. The method according to any one of the preceding claims, wherein the step of recovering said cyclic peptides, polypeptides or proteins from said cells or said medium of said cell or tissue culture, or both, further comprises the step of inducing a permeabilisation of the cell membranes of the cells of said cell or tissue culture.

22. The method according to any one of the preceding claims for the production of circulins, cycloviolins , cycloviolacin, kalatas, cyclopsychotrids, palicoureins, vitri, vodo, vico, varv peptides, viola peptides hypa A or McoTO .

23. Device for carrying out a process according to the invention, comprising a bioreactor (101) further comprising a plant cell or tissue culture derived from a Viola species, Cucurbitaceae species, Rubiaceae species and

1) means for the extraction of a peptide, polypeptide or proteine suspension from the bioreactor (101) 2) an irradiation source (102) capable of emitting visible light with an energy density in the range of 5 - 30 μEm^"2s^_1.

24. A device according to claim 23, wherein the plant cell or tissue culture is derived from Chassalia species, Oldenlandia species, Psychotria species, Palicourea species, Leonia species, Hybanthus species or Momordica species .

25. A device according to claim 23 or 24, wherein the bioreactor comprises a recombinant or a non-recombinant plant cell or tissue culture.

26. A device according to one of the preceding claims, wherein the bioreactor comprises a suspension culture, a hairy root culture or an embryonic culture.