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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
• • C—CHEMISTRY; METALLURGY
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  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
  • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
  • C12N5/0025—Culture media for plant cell or plant tissue culture
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/04—Plant cells or tissues

Definitions

• This invention relates to plant-derived secondary metabolites and, more particularly, to a method and means for inducing plant cells to produce these metabolites.
• a number of chemicals produced by plants are classified as primary metabolites since they are essential to cell function. While primary metabolites are ubiquitous in the plant population and comprise the majority of plant-produced chemicals, other compounds are classified as secondary metabolites and, because of their various applications, are the focus of both scientific investigation and commercial exploitation.
• secondary metabolites are produced by some plants in response to stress induced, for example, by microorganism infection, UV irradiation, mechanical wounding as well as by treatment with organic and inorganic substances.
• the variety of secondary metabolites produced under such stressful conditions is diverse and includes the alkaloids, terpenoids, flavonoids, carotenoids, phenolic compounds and glucosides.
• pharmaceutically useful secondary metabolites are the alkaloids, steroid hormones, cardiac glycosides and antibiotics, of which the indolic alkaloids are of particularly great interest.
• the alkaloids include many compounds, at least some of which are known to have therapeutic value.
• the indolic alkaloid ajmalicine is known to function as an antihypertensive/tranquilizer.
• vincristine and vinblastine are accepted in the treatment of cancers.
• Catharanthine a precursor of both vinblastine and vincristine, is also produced as a secondary metabolite and is valuable as a starting material in preparing those compounds.
• metabolite produced depends largely on the plant species. For example, some indolic alkaloids are extracted most commonly from Catharanthus roseus (also known as Madagascar periwinkle, Vinea rosea and Lochnera rosea). Further, morphinan alkaloids e.g. morphine and codeine, are extracted from poppy plants (Papaver somniferum) and Cinchona alkaloids e.g. quinine and related quinoline alkaloids, are derived from Cinchona succirubra and related species. Obviously, the plant from which the secondary metabolite is to be derived must be genetically capable of expressing the metabolite and must be receptive to a particular metabolic stimulus in order to engage in synthesis of the metabolite.
• Plant tissue culture entails growth of a population of plant cells suspended within nutrient broth using a level agitation which strikes a balance between the need to circulate nutrients throughout the culture and the fragility of the plant cells. Not all plants are amenable to this procedure, however, and the media constitution is also a major factor in the success of the method.
• Advantages of the ptc technique reside in the ability to exercise control over the growth conditions in terms of defined environment, nutrient concentration, and the like. Further, the capacity to circulate a stimulant within the culture broth allows the stimulant to contact a greater number of the individual cells or cell clumps in the broth by comparison with application of the stimulant to a callus whereby only the outermost cells may be effected.
• Eilert et al. disclose an attempt to induce production of the antimicrobial alkaloids rutacridone epoxide and hydroxyrutacridone epoxide from a suspension culture of Ruta graveolens.
• a suspension of either living free or immobilized yeasts or of dead Rhodotorula rubra cells or a crude cell wall fraction thereof they were able to show an increase in production of the antimicrobial compounds of interest.
• neither chitosan nor alginate were able to induce the desired response despite their having been proven to be useful as elicitors in other systems.
• the stress caused by the varied osmotic pressure is believed to be responsible for the increased synthesis.
• Those compounds which may be added to the plant cell suspension to generate osmotic stress may be selected from a very broad range of compounds. In general, these compounds may be categorized as ionic stress inducing compounds and organic stress inducing compounds. In either case, the end result of their addition to a suspension is the creation of an osmotic gradient between the cell and the surrounding medium.
• the ionic, stress-inducing compounds are preferably those halide salts able to ionize in solution. More preferably these salts are Group I or Group II halide salts, the Group numbers referring to the Periodic Table. Especially suitable such compounds include the Group I and Group II chlorides although the bromide salts are also suitable. In a limited sense, the invention extends to such compounds as the chloride salts of sodium, potassium and iron (preferably ferric).
• the organic, stress-inducing compounds include the sugar alcohols and sugar acids which include galactinol, xylitol, glycerol, mannitol and inositol and its various derivatives including phosphatidyl inositol, phytie acid and its esters, scyllitol, phytol, aldonic acids, aldaric acids, uronic acids and, notably, sorbitol. Sorbitol in particular has induced significant yields of secondary metabolites and is therefore preferred. Also within the scope of the present invention are compounds which act to induce secondary metabolite production by means other than through osmotic stress.
• This aspect of the present invention comprises culturing plant cells in a suspension supplemented with any one or a combination of a variety of plant growth regulating compounds and compounds which are related either by chemical structure or be biochemical function. While abscissic acid (ABA) is particularly useful, other related compounds which may be used include the antigibberellin compounds such as 2'-isopropyl-4'-(tri- methylammonium chloride)-5'-methylphenyl piperidine carboxylate, ⁇ -chloroethyltrimethylammonium chloride and tributyl-2,4-dichlorobenzyl phosphonium chloride.
• ABA abscissic acid
• other related compounds which may be used include the antigibberellin compounds such as 2'-isopropyl-4'-(tri- methylammonium chloride)-5'-methylphenyl piperidine carboxylate, ⁇ -chloroethyltrimethylammonium chloride and tributyl
• ABA-related compounds useful herein include N- (dimethyl- amino) succinamic acid, 4 ' -dihydrophaseic acid, phaseic acid and lunularic acid.
• Another aspect of the invention comprises a process for inducing synthesis of a secondary metabolite by plant cells which comprises growing said cells in the presence of a plant growth regulating compound or related such compound.
• the plant cells are cultured in a suspension supplemented with the regulator.
• the desired secondary metabolite or metabolites may be recovered using chemical procedures currently established in the biochemical extraction art.
• metabolites may simply be separated from the cells per se by concentrating the cells using conventional techniques such as centrifugation and filtration. Thereafter, the broth containing the metabolite may either be processed further to concentrate the metabolite by such techniques as dialysis or acted upon directly using standard chemical extraction techniques. Where the desired metabolite is expressed by the plant cell but is not secreted from it, the plant cells are broken open using, for example, increased pressure, either atmospheric or osmotic, or crushed. The metabolite may then be extracted in a suitable solvent and concentrated somewhat using procedures outlined above prior to chemical extraction of the desired secondary metabolite.
• a second aspect of the present invention comprises a method of providing a plant-cell derived secondary metabolite which comprises recovering the compound produced by way of the induction process of the present invention.
• the indolic alkaloids obtainable from Catharanthus roseus including NYCoganin and tryptophan-derived strictosidine, ajmalicine, yohimbine, tabersonine, vindoline and catharanthine as well as tryptamine; tyrosine-derived morphine and codeine from Papaver somniferum; phenylalanine-derived chemicals such as coumarins from parsley cell cultures; and mevalonic acid-derived chemicals such as saponins e.g. digitalis from Digitalis purpurea or D. lanata.
• the present invention related in a preferred aspect to the C . roseus derived indole alkaloids catharanthine, ajmalicine, tabersonine and vindoline.
• the inducing substance abscissic acid used herein is a known compound having the structural formula appearing below:
• Abscisic acid is an abscission-accelerating plant hormone i.e. a hormone which promotes separation of plant parts such as leaves from stems during the autumn season. It is a commercially available commodity known also as dormin whose commercial nomenclature is 5-(1hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl)-3- methyl-2,4-pentadienoic acid. Synthetic forms of the drug as well as various cis-trans isomeric forms are also available commercially and may be used herein.
• plant cell culturing techniques are known and are used preferably herein.
• Conventional procedures are therefore employed in which living plant material e.g. leaf material, stem material or meristems are surface sterilized to prevent contamination and smaller masses of individual cells or cell clumps are nursed on agar nutrient base in growth plates until they may be transferred to a suitable liquid nutrient.
• the suspension is agitated continuously in culture flasks and ultimately transferred to a bioreactor. Agitation is suitably accomplished by the "air-lift" technique in which the culture is mixed by the gentle action of rising air bubbles.
• a solution of the inducing substance is suitably pre-prepared if desired in order to enhance homogeneity of the substance within the growth-sustaining medium.
• all substances of the present invention may be mixed with water prior to addition.
• Abscisic acid may be mixed with water and a few drops of base such as KOH to encourage dissolution if necessary.
• the preferred embodiment of the invention provides a method by which indolic alkaloids are produced by Catharanthus roseus.
• the C. roseus line from which the cultured cells are derived must, obviously, be amenable to plant tissue culturing. Obtaining such a line is well within the skill of persons to whom this disclosure is addressed and it is believed that several such lines presently exist. This is not to detract from the need to provide a plant whose cells have the capacity to endure and function within the cell culture environment. Those characteristics which confer this capacity are not well defined in the art. The presence of cells which are plant tissue culturable is confirmed almost exclusively by the trial and error approach. Thus, while specific cell lines having designations assigned by the inventors is used to exemplify the present invention hereinafter, it is to be acknowledged that the scope of the invention is by no means limited to these particular plant lines.
• C. roseus lines herein are designated JWM*, JOH and LBE-1.
• Lines JWM* and JOH were developed at the Plant Biotechnology Institute, Saskatoon, Sasketchewan, Canada.
• Line LBE-1 was developed at Allelix Inc., Mississauga, Ontario, Canada. All lines were originally isolated from C. roseus anthers.
• the C. roseus cells may be cultured in any liquid medium containing all necessary metabolites, examples of which include SH medium, LS medium and MS medium, all of which are known in the art.
• LS medium Lismair and Skoog
• SH medium Shinek-Hildebrandt
• MS medium Murashige and Skoog
• a carbon source such as sucrose or lactose.
• the sole growth regulator added to the SH medium is ⁇ -naphthaleneacetic acid, in a concentration of 2 mg/1.
• the medium also suitably contains kinetin (6-furfurylaminopurine) which is a synthetic cytokinin which functions in cell division.
• the plant cells are grown either in the dark to reduce stress and channel nutrients from light-activated secondary metabolic pathways (e.g. pigment formation) or light preferably until the population reaches the linear or early stationary growth phase at which time they are presumed to be more receptive to the inducing effects of the selected inducing preparation.
• the C. roseus cell suspension is grown in the absence of inducer preferably for from 3 to 10 days, more preferably for from 4 to 6 days under the conditions tested.
• the cell population may be monitored in order to identify the onset of the linear or early stationary growth phase if alternative growth conditions are employed.
• the inducing substance is introduced in amounts sufficient to establish a desired concentration within the broth.
• the inducing substances preferred herein are NaCl, KCl and FeCl 3 as ionic, osmotic stress inducers; sorbitol as organic, osmotic stress inducer and abscissic acid (ABA) as plant growth regulator inducer.
• the final broth concentration of either NaCl or KCl is suitably about 0.1 M, as a minimum desirable level. Lower levels may serve the inducing purpose but are unlikely to provide enhanced yields within the shortened time periods available with higher concentrations.
• the acceptable upper concentration level of NaCl or KCl is dictated by the osmotic pressure which its presence generates within the cultured cells. The upper limit is therefore slightly below that concentration which causes plasmolysis. More preferably, NaCl and KCl may be added to achieve broth concentrations of 0.01 M to 1.0 M and, ideally, at around 0.5 M. Ferric chloride is suitably added to the suspension medium to achieve a final concentration of from 10 - 500 ppm, more preferably from 20 - 200 ppm and, ideally, around 50 ppm.
• Broth concentrations of sorbitol range from about 0.05 M to a maximum concentration just slightly below the concentration at which plasmolysis occurs, as discussed with respect to NaCl or KCl addition.
• Preferred broth concentrations range from 0.1 M to 0.5 M and ideally at around 0.2 M.
• the ideal concentration of abscissic acid ranges from 0.1 mg- 0.5 mg per 60 mis of suspension. Concentrations as low as 0.01 mg may be used although the induction response may be unfavourably low. Concentrations higher than 0.5 mg/60 mls also may be used although there appears to be little enhancement of the effect seen at 0.5 mg/60 mis.
• the C. roseus cells are cultured for a period of time to allow the inducer to interface with the cells and stimulate indolic alkaloid production.
• a period of 2 - 5 days is preferred although variations of this preferred period are acceptable particularly where conditions are not as specifically described herein. Growth of the culture is preferably continued either in the dark or in light.
• the cells are harvested and the alkaloids extracted using conventional techniques. The particular techniques will depend on the specific substance to be recovered, but all are standard in the art.
• Embodiments of the invention are disclosed hereinafter with reference to the following examples.
• 60 ml cell suspension cultures are maintained in a basic MS growth medium containing 3% (w/v) sucrose, 2 mg/l ⁇ -naphthalenic acid and 0.1 mg kinetin.
• the cultures are maintained in 250 ml Erlenmeyer flasks on an illuminated rotary shaker (c. 120 rpm) and subcultured weekly by a one to five dilution.
• Abscisic acid and D-sorbitol were obtained from Sigma Chemical Company and NaCl, KCl and FeCl 3 from Fisher Scientific Limited.
• each inducer was added to the cell suspension on day 5 of the growth cycle as a filter-sterilized solution and the cells harvested three days later. Standard solvent extraction techniques were used to extract indolic alkaloids from the cells. Catharanthine and ajmalicine yields were quantified by HPLC analysis. The presence of other alkaloids was determined qualitatively by visualizing with eerie ammonium sulphate following TLC separation.
• Example 1 - ABA Induction Synthesic 99% pure abscissic acid was introduced in a range of concentrations from 0.1 - 2.0 mg/60 ml culture. The results generated appear in Tables 1 - 4 below. Table 1 shows the effect which the type and concentration of ABA has on the induction process as applied to cell line JOH. While the 99% pure ( ⁇ )cistrans synthetic isomer exhibited superior results, all forms of ABA showed desirable inducing properties.
• Table 2 illustrates the effect of ABA concentration on induction response on line JWM*. It will be noted that a maximum response occurs at 0.5 mg/60 ml culture with only minor variation with increased concentration.
• Table 3 the relationship of cell age and exposure to ABA is shown. It will be noted that atleast 3 days of culture growth provided the best results, with ajmalicine concentrations dropping off thereafter. However, catharanthine yields continue to rise even at day 6 of culture growth. The Table 3 results are with cell line JOH and an induction period of 4 days.
• Table 4 below shows the response to 1 mg ABA induction of a variety of cell lines each of which is available at either Allelix Inc. or the Plant Biology
• the plant cells have been observed to respond positively to ABA exposure by increased alkaloid production even after only one day of growth, when they are presumably still in lag phase. However, as alkaloid accumulation is to some extent biomass related, it is more appropriate to induce the cells once some increase in biomass has occurred.
• Example 2 Induction by NaCl
• a range of sodium chloride concentrations (from 0.1 - 1.0 g/60 ml flask) were tested for their effects on alkaloid accumulation in line JOH Table 5 below. High concentrations of NaCl reduce biomass, hence lowering alkaloid yields, whereas the effect on biomass of lower concentrations is offset by the stimulation of catharanthine production.
• Example 5 Induction by Sorbitol
• the major advantage obtained by inducing cell culture in accordance with the present invention is that yields of alkaloids (specifically catharanthine) can reach levels in only 8 days that in non-induced cells would take much longer to achieve, resulting in a considerably saving in terms of both time and cost.
• alkaloids specifically catharanthine
• the MS medium is enhanced preferably with 4% (w/v) sucrose as opposed to the 3% solution used on small scale, together with 1 mg/l ct-naphthalene acetic acid and 0.1 mg/l kinetin. All lines and chemicals remain the same.
• Example 6 Larger Scale Induction Process
• solutions are preferably prepared to give 8.33 mg/1 in the final culture fluid.
• Solutions are prepared in 1 liter of distilled water, filter sterilized and injected into the fermenter from a sterile bottle.
• Solutions of NaCl are prepared in 1 liter of distilled water to give a final concentration in the culture of 33.33 g/l. Solutions are filter sterilized and added to the culture from a sterile bottle.
• Inductions usually take place on day 5 to 7 of a culture period. Stimulations usually occur within 24 hours and can continue for up to 7 days.
• Synthesis of alkaloids by suspension cultured Catharanthus roseus cells is enhanced by stressing the cultured c osmotically by addition to the culture of such inducers as ionic osmotic stressors such as halide salts e.g. NaCl, KCl FeCl 3 or organic osmotic stressors such as sugar alcohols and sugar acids.
• ionic osmotic stressors such as halide salts e.g. NaCl, KCl FeCl 3 or organic osmotic stressors such as sugar alcohols and sugar acids.
• Synthesis of alkaloids is also enhanced by a tion of anti-gibberellin compounds and other compounds such as abscissic acid which is a plant growth regulator. Yi of alkaloids are consequently increased.

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• 1987
  • 1987-08-03 EP EP87904973A patent/EP0277250A1/en not_active Withdrawn
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