Classifications

• • 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
  • C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings

Definitions

• This invention relates to a method for mass production of secondary
• metabolites in plant cell culture and more specifically, to a method for production of secondary metabolites by using plant cell culture, characterized in that a faster growth of plant cells and the enhanced productivity of secondary
• metabolites are achieved in plant cell culture by treating culture medium with an alkanoic acid or salt thereof.
• the culture environments such as temperature, lighting, pH of the medium,
• genes relating to defense mechanisms while at the same time repressing the expression of genes relating to cell cycle control such as p34cdc2 protein kinase and mitotic cyclin (Logemann et al., Plant Journal 8:865-876, 1995).
• An objective of the present invention is to provide a method for mass production of secondary metabolites in plant cell cultures. Another objective of the present invention is to provide optimal conditions in the treatment concentrations, time of treatment, and the number of repeated treatments of alkanoic acid or salt thereof to enhance the productivity of secondary metabolites in plant cell cultures.
• the present invention provides a method for production of secondary metabolites by plant cell culture which includes the step of culturing plant cells upon treatment of the culture
• the present invention provides a method for mass production of secondary metabolites from plant cells by using alkanoic acid or salt thereof
• metabolites of plant origin such as paclitaxel, corosolic acid and the like.
• Fig. 1 shows time course of changes in dry cell weight of Taxus
• chinensis cell line SYG-1 after treating with 0 to10 mM of sodium butyrate on
• Fig. 2 shows the production pattern of paclitaxel in Taxus chinensis
• FIG. 3 is a photograph showing an electrophoresis result of the
• Fig. 4 shows time course of changes in the change in the dry cell
• Taxus chinensis cell line SYG-1 after culture following treatment with
• Fig. 5 shows the production pattern of paclitaxel after treating the
• Fig. 6 shows the result of paclitaxel production from Taxus chinensis
• Fig. 7 shows taxane produced from Taxus chinensis cell line SYG-1
• Fig. 8 shows tome course of changes in dry cell weight of Taxus
• chinensis cell line SYG-1 after treating with sodium propionate.
• Fig. 9 shows the result of paclitaxel production from Taxus chinensis
• Fig. 10 shows the result of paclitaxel production from Taxus chinensis
• SF sodium acetate
• SP sodium propionate
• SB sodium butyrate
• FIG. 11 and 12 show HPLC charts of suspension culture solution of
• concentration of alkanoic acid or salt thereof enhances cell growth, and found the optimal conditions for treating with alkanoic acid or salt thereof to produce secondary metabolites in plant cell cultures in mass quantities.
• secondary metabolites There are a wide variety of secondary metabolites that can be
• Table 2 shows examples of the above secondary metabolites and plant cells that can be used in production thereof.
• the method in accordance with the present invention for mass production of secondary metabolites in plant cell culture can be applied to all plant cells that produce secondary metabolites.
• the method can enhance productivity of secondary metabolites by applysing it to various plant cells displaying low productivity of secondary metabolites, and in particular be used in commercial production of paclitaxel by applying to Taxus genus cells
• paclitaxel used to produce paclitaxel which is proven to be effective in treatment of treatment-resistant ovarian cancer and breast cancer.
• Taxus cells belonging to the Taxus genus can have enhanced productivity of secondary metabolites according to the
• Taxus genus includes, but is not limited
• Taxus bacata Taxus brevifolia
• Taxus canadensis Taxus chinensis
• Taxus chinensis Taxus
• Taxus flo dana cuspidata, Taxus flo dana, Taxus globosa, Taxus media, Taxus wallichiana,
• Taxus yunnanensis Mass production of paclitaxel and taxane from plant cells
• a method for mass production of secondary metabolites in plant cell cultures according to the present invention includes the step of treating the plant cell culture with at least one alkanoic acid or salt thereof. .
• the above plant cell culture As it is widely known in the related art, the above plant cell culture
• plant cell culture media can comprise nutrients and other factors necessary to maintain the viability of plant cells, that is, carbon sources, nitrogen sources, salts, vitamins and the like.
• the plant cell culture media of the present invention can be media widely used in culturing plant cells, for example, but not limited to
• B5 medium comprising casein hydrolysate (see:
• a plant cell culture medium according to the present invention comprises selectively factors that can induce the production of secondary metabolites.
• factors include plant hormones, biosynthetic precursors or elicitors and signal couplers of secondary metabolites and the like. These factors added in the medium stimulate the production of secondary metabolites.
• the preferred inducing agent is silver
• nitrate and is preferably present in the culturing medium in the concentration range between 1uM and 15uM and added to the medium in the early stages of
• culture methods and conditions can be selected among
• Alkanoic acid according to the present invention is a compound linked
• n can be an integer from 1 to 9, preferably from 3 to 6.
• an alkanoic acid can be an alkaline metal salt, for example, sodium (Na), potassium (K) and the like are suitable.
• salt of butyric acid or salt of propionic acid preferably sodium butyrate or sodium propionate can be used.
• Alkanoic acids or salts thereof play a role in controlling the plant cell cycle by inducing modification of chromosome structure in cells by increasing
• Alkanoic acid or salt thereof may be present in culture medium at a concentration of between 0.01 to 500 mM, preferably between 0.1 mM to 200 mM, and more preferably between 0.1 mM to 20 mM, but is most preferably to
• Taxus chinensis cell growth is enhanced on culturing
• paclitaxel is increased significantly to 1.97 times that of control (Fig. 2).
• the treatment time of alkanoic acid or salt thereof to culture medium can be from prior to inoculation of the culture medium with plant cells to the end of growth and activity of the cells, and preferably the early log growth phase of the cells. It is more preferable, however, to adjust the time of treatment with alkanoic acid or salt thereof depending on plant cell type.
• the medium may be
• alkanoic acid or salt thereof from day 0 to day 21 of culture, and more preferably from day 0 to day 14 of culture.
• Alkanoic acid or salt thereof induces physiological changes of plant cells, and particularly induces changes such as inhibition of histone deacetylase activity or suppression of cell cycle related gene expression.
• the alkanoic acid or salt thereof is present in the medium at a constant concentration. Therefore, after the initial treatment of the culture medium with alkanoic acid or salt thereof, the culture medium may be treated one or more times at regular intervals. And alkanoic acid or salt
• concentration of the alkanoic acid or salt thereof be varied having regard to the
• Taxus chinensis cells can be treated with alkanoic acid or
• a method of the present invention can be applied to increase the
• Type II diabetes by Eriobotrya japonica cell culture.
• the present invention provides plant cell culture media and media for the production of secondary metabolites from plant cell culture
• alkanoic acid or salt thereof may be any suitable alkanoic acid or salt thereof.
• the alkanoic acid or salt thereof may be any suitable alkanoic acid or salt thereof.
• the concentration of the alkanoic acid or salt thereof may be appropriately varied having regard to the
• EXAMPLE 1 The effect of treating plant cell cultures with sodium
• Taxus chinensis SYG-1 cell line (KCTC-0232BP) was used to produce paclitaxel and taxane compounds.
• butyrate was added at concentrations of 0, 0.5, 1 , 5 and 10 mM, respectively
• cell weight is weight of cells determined after drying in a drying oven at 60 ° C
• lane A is a size marker
• lane B is a non-treated sample
• lane C is a sample treated with 1 mM of
• sodium butyrate and lane D is a sample treated with 5 mM of sodium butyrate.
• EXAMPLE 2 Determination of time of sodium butyrate treatment Sodium butyrate was added at concentrations of 1 mM on day 0, day
• Fig. 4 shows the cell growth according to the treatment time of sodium
• FIG. 5 shows paclitaxel production (in the Fig. 4 and Fig. 5 - ⁇ :
• non-treated sample o: sample treated with 1 mM of sodium butyrate on day 0
• paclitaxel productivity was highest when 1 mM of sodium butyrate was treated
• EXAMPLE 3 Repetitive treatment of sodium butyrate While culturing Taxus chinensis SYG-1 cell by using the same
• Taxus chinensis SYG-1 cell While culturing Taxus chinensis SYG-1 cell by using the same method as in EXAMPLE 1-1 , 1 Mm of sodium butyrate was added on day 7 and day 14 of culture. The culture was sampled on day 28, day 36 and day 43 of culture in order to determine the productivity of taxane compounds.
• Fig. 7 shows the productivity of taxane compounds (rate of increase
• butyrate on secondary metabolite productivity increase is a kind of induction
• Taxus chinensis SYG-1 cells While culturing Taxus chinensis SYG-1 cells, 0.5 mM or 1 Mm of
• Fig. 8 shows the cell growth of SYG-1 cultured with sodium
• Fig. 9 shows the productivity of paclitaxel (in the Fig.
• sodium propionate was 1 mM and when treated two times repeatedly.
• Fig. 10 shows paclitaxel productivity of Taxus chinensis SYG-1 cells
• SA sodium propionate
• SB sodium butyrate
• VA valeric acid
• induced callus was transferred to agar-free liquid culture medium and suspension cultured at 24 °C, 120 rpm in the dark.
• the established suspension cultured cells of Eriobotrya japonica was subcultured in the above medium at two-week intervals.
• the suspension culture cell of Eriobotrya japonica was treated with
• Fig. 11 and 12 show HPLC charts of extracts from Eriobotrya japonica cells cultures in the absence(Fig. 11) and the presence(Fig. 12) of 0.5 mM of

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• 2004
  • 2004-12-29 WO PCT/KR2004/003491 patent/WO2005064002A1/en not_active Application Discontinuation
  • 2004-12-29 EP EP04808620A patent/EP1709185A1/de not_active Withdrawn

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