Enhanced production of taxol and taxanes by cell cultures of taxus species

Abstract

This invention provides methods whereby taxol, baccatin III, and other taxol-like compounds, or taxanes, can be produced in very high yield from all known Taxus species, e.g., brevifolia, canadensis, cuspidata, baccata, globosa, floridana, wallichiana, media and chinensis. Particular modifications of culture conditions (i.e., media composition and operating modes) have been discovered to enhance the yield of various taxanes from cell culture of all species of Taxus. Particularly preferred enhancement agents include silver ion or complex, jasmonic acid (especially the methyl ester), auxin-related growth regulators, and inhibitors of the phenylpropanoid pathway, such as 3,4-methylenedioxy-6-nitrocinnamic acid. These enhancement agents may be used alone or in combination with one another or other yield-enhancing conditions. While the yield of taxanes from plant cell culture of T. chinensis is particularly enhanced by use of one or more of these conditions, yield of taxanes for all Taxus species has been found to benefit from use of these conditions.

1 . A method for producing one or more taxol and other taxanes in high yields in cell culture of a  Taxus  species comprising: cultivating in suspension culture, in one or more nutrient media under growth and product formation conditions, cells of a  Taxus  species derived from callus or suspension cultures, and recovering said one or more taxol or other taxanes from said cells, said medium of said cell culture, or both, wherein at least one of the one or more nutrient media comprises an enhancement agent selected from the group consisting of (a) jasmonate-related compound or an alkyl ester thereof, (b) an antiethylene agent, (c) an inhibitor of phenylpropanoid metabolism, (d) a polyamine, and (e) β-phenylalanine.  
     
     
         2 - 62 . (canceled)  
     
     
         63 . A method for producing taxol and other taxanes from cell culture of a  Taxus  species comprising: cultivating in suspension culture, in a first medium developed for a growth phase and then in a second medium developed for a product formation phase, cells of a  Taxus  species derived from callus and/or suspension cultures, and recovering said taxol or other taxanes from said cells, said first medium and/or said second medium of said cell culture.  
     
     
         64 . The method of  claim 63 , wherein at least one of said first medium and said second medium comprises a polyamine.  
     
     
         65 . The method of  claim 64 , wherein the polyamine is selected from the group consisting of spermine, spermidine, putrescine, cadaverine, and diaminopropane.  
     
     
         66 . The method of  claim 63 , wherein at least one of said first medium and said second medium comprise a taxane precursor.  
     
     
         67 . The method of  claim 63 , wherein the concentration of nitrate is lower in the second medium than in the first medium, and the concentration of saccharide is higher in the second medium than in the first medium.  
     
     
         68 . The method of  claim 67 , wherein the first medium contains nitrate at a concentration which is 2 to 10 times the nitrate concentration in the second medium.  
     
     
         69 . The method of  claim 67 , wherein the second medium contains saccharide at a concentration which is 2 to 5 times the saccharide concentration in the first medium.  
     
     
         70 . The method of  claim 67 , wherein the first medium contains saccharide in the concentration of 1-30 g/L, and nitrate ion in the concentration of 2.5-70 mM; and the second medium contains saccharide in the concentration of 4-150 g/L, and nitrate ion in the concentration of 0.3-18 mM.  
     
     
         71 . The method of  claim 67 , wherein the first medium contains saccharide in the concentration of 5-15 g/L, and nitrate ion in the concentration of 20-30 mM; and the second medium contains saccharide in the concentration of 35-55 g/L, and nitrate ion in the concentration of 2-7 mM.  
     
     
         72 . The method of  claim 63 , wherein the medium is replenished during cultivation by periodically replenishing nutrient medium components and removing spent medium.  
     
     
         73 . The method of  claim 63 , further comprising the periodic removal of said taxol and other taxanes from said medium.  
     
     
         74 . The method of  claim 63 , wherein the  Taxus  species is selected from the group consisting of  T. canadensis, T. chinensis, T. cuspidata, T. baccata, T. globosa, T. floridana, T wallichiana,  and  T. media.    
     
     
         75 . The method of  claim 63 , wherein said step of cultivating is carried out in the presence of 0.03% to 15% v/v of carbon dioxide in the gas phase in equilibrium with at least one of said first medium and said second medium.  
     
     
         76 . The method of  claim 63 , wherein said step of cultivating is carried out in the presence of 0.3% to 8% v/v of carbon dioxide in the gas phase in equilibrium with at least one of said first medium and said second medium.  
     
     
         77 . The method of  claim 63 , wherein said step of cultivating is carried out in the presence of dissolved oxygen concentration between 1% to 200% v/v of air saturation.  
     
     
         78 . The method of  claim 63 , wherein said step of cultivating is carried out in the presence of dissolved oxygen concentration between 10% to 100% v/v of air saturation.  
     
     
         79 . The method of  claim 63 , wherein said step of cultivating is carried out in the presence of dissolved oxygen concentration between 25% to 95% v/v of air saturation.  
     
     
         80 . The method of  claim 63 , wherein at least one of said first medium and said second medium comprises glutamine.  
     
     
         81 . The method of  claim 64 , wherein said polyamine is added in an amount sufficient to enhance taxane production.  
     
     
         82 . The method of  claim 63 , wherein β-phenylalanine is added to at least one of said first medium and said second medium in an amount sufficient to enhance taxane production.  
     
     
         83 . The method of  claim 63 , wherein at least one of said first medium and said second medium comprises one or more enhancement agents selected from the group consisting of (a) jasmonate-related compounds or alkyl esters thereof, (b) antiethylene agents, and (c) inhibitors of phenylpropanoid metabolism.  
     
     
         84 . The method of  claim 83 , wherein at least one of said first medium and said second medium comprises an antiethylene agent which is a silver-containing compound, or a silver complex, or a silver ion.  
     
     
         85 . The method of  claim 83 , wherein the jasmonate-related compound or an alkyl ester thereof is added to at least one of said first medium and said second medium.  
     
     
         86 . The method of  claim 85 , wherein the jasmonate-related compound is at least one compound selected from the group consisting of jasmonic acid and dihydrojasmonic acid.  
     
     
         87 . The method of  claim 85 , wherein the jasmonate-related compound is at least one compound selected from the group consisting of jasmonic acid and alkyl esters of jasmonic acid.  
     
     
         88 . The method of  claim 87 , wherein the jasmonic acid alkyl ester is methyl jasmonate.  
     
     
         89 . The method of  claim 83 , wherein at least one of said first medium and said second medium comprises an inhibitor of phenylpropanoid metabolism.  
     
     
         90 . The method of  claim 63 , wherein at least one of said first medium and said second medium comprises an auxin-related growth regulator selected from the group consisting of 1-Naphthaleneacetic acid, 2-Naphthaleneacetic acid, 1-Naphthaleneacetamide/Naphthylacetamide, N-(1-Naphthyl)phthalamic acid, 1-Naphthoxyacetic acid, 2-Naphthoxyacetic acid, beta-Naphthoxyacetic acid, 1-Naphthoxyacetamide, 3-Chlorophenoxyacetic acid, 4-Chlorophenoxyacetic acid, 4-Iodophenoxyacetic acid, Indoleacetamide, Indoleacetic acid, Indoylacetate, Indoleacetyl leucine, Gamma-(3-Indole)butyric acid, 4-Amino-3,5,6-trichloropicolinic acid, 4-Amino-3,5,6-trichloropicolinic acid methyl ester, 3,6-Dichloro-o-anisic acid, 3,7-Dichloro-8-quinolinecarboxylic acid, Phenylacetic acid, 2-Iodophenylacetic acid, 3-Iodophenylacetic acid, 2-Methoxyphenylacetic acid, Chlorpropham, 4-chloroindole-3-acetic acid, 5-Chloroindole-3-acetic acid, 5-Bromo-4-chloro-3-indoyl butyrate, Indoleacetyl phenylalanine, Indoleacetyl glycine, Indoleacetyl alanine, 4-chloroindole, p-chlorophenoxyisobutyric acid, 1-pyrenoxylbenzoic acid, Lysophosphatidic acid, 1-naphthyl-N-methylcarbamate, Ethyl-5-chloro-1H-Indazole-3-ylacetate-3-Indolebutanoic acid, Naphthalene-2,6-dicarboxylic acid, Naphthalene-1,4,5,8-tetracarboxylic acid dianhydride, Naphathalene-2-sulfonamide, 4-Amino-3,6-disulfo-1,8-naphthalic anhydride, 3,5-dimethylphenoxyacetic acid, 1,8-Naphthalimide, 2,4-Dichlorophenoxyacetic acid, 2,3-Dichlorophenoxyacetic acid, 2,3,5-Trichlorophenoxyacetic acid, 2-Methyl-4-chlorophenoxyacetic acid, Nitrophenoxyacetic acids, DL-alpha-(2,4-Dichlorophenoxy)propionic acid, D-alpha-(2,4-Dichlorophenoxy)propionic acid, 4-Bromophenoxyacetic acid, 4-Fluorophenoxyacetic acid, 2-Hydroxyphenoxyacetic acid, 5-Chloroindole, 6-Chloro-3-indoylacetate, 5-Fluoroindole, 5-Chloroindole-2-carboxylic acid, 3-Chloroindole-2-carboxylic acid, Indole-3-pyruvic acid, 5-Bromo-4-chloro-3-indoylbutyrate, 6-Chloro-3-indoylbutyrate, Quinoline-2-thioglycolic acid, Aminophenylacetic acids, 3-Nitrophenylacetic acid, 3-Chloro-4-hydroxybenzoic acid, Chlorflurenol, 6-Chloro-3-indoyl acetate, N-(6-aminohexyl)-5-chloro-1-Naphthalenesulfonamide hydrochloride, 2-chloro-3(2,3-dichloro-phenyl)propionitrile, o-chlorophenoxyacetic acid, 6,7-dimethoxy-1,2-benzisoxazole-3-acetic acid, 3-oxo-1,2,-benzisothiazoline-2-ylacetic acid, Mastoparan, 2,3,5-Triidobenzoic acid, 2-(3-chlorophenoxy)propanoic acid, Mecoprop, Naphthoic acid hydrazide, 2,4-Dibromophenoxyacetic acid, 3-Trifluoromethylphenoxyacetic acid, Oxindole, Indole-2-carboxylic acid, Indole-3-lactic acid, Beta-(3-Indole)propionic acid, 2-Bromophenylacetic acid, 3-Bromophenylacetic acid, 2-Chlorophenylacetic acid, 3-Chlorophenylacetic acid, 2-Methylphenylacetic acid, 3-Methylphenylacetic acid, 3-Trifluoromethylphenylacetic acid, 3-Methylthiophenylacetic acid, Phenylpropionic acid, 4-chloro-2-methylphenylthioacetic acid, 2-Chlorobenzoic acid, 3-Chlorobenzoic acid, 2,3-Dichlorobenzoic acid, 3,4-Dichlorobenzoic acid, 2,3,5-Trichlorobenzoic acid, 2,4,6-Trichlorobenzoic acid, 2-Benzothiazoleoxyacetic acid, 2-Chloro-3-(2,3-dichlorophenyl)propionitrile, 2,4-Diamino-s-triazine, Naphthalic anhydride, Dikegulac, chlorflurecolmethyl ester, 2-(p-chlorophenoxy)-2-methylpropionic acid, 2-chloro-9-hydroxyfluorene-9-carboxylic acid, 2,4,6-trichlorophenoxyacetic acid, 2-(p-chlorophenoxy)-2-methyl propionic acid, Ethyl 4-(chloro-o-tolyloxy)butyrate, [N-(1,3-dimethyl-1H-Pyrazol-5-yl)-2-(3,5,6-Trichloro-2-pyridinyl)oxy]acet-amide, 4-Chloro-2-oxobenzothiazolin-3-yl-acetic acid, 2-(2,4-Dichlorophenoxy)propanoic acid, 2-(2,4,5-Trichlorophenoxy)propanoic acid, 4-Fluorophenylacetic acid, 3-Hydroxyphenylacetic acid, Orthonil, 3,4,5-Trimethoxycinnamic acid, 2(3,4-dichlorophenoxy)triethylamnine, Indole-3-propionic acid, Sodium Ioxynil, 2-Benzothiazoleacetic acid, and (3-phenyl-1,2,4-thiadiazol-5-yl)thioacetic acid.