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.

Description

[0001] This application is a continuation-in-part of International application PCT / US97 / 08907, designating the U.S. filed May 27, 1997, and a continuation-in-part of U.S. Ser. No. 08 / 653,036, filed May 24, 1996, which is a continuation-in-part of U.S. Ser. No. 08 / 370,494, filed Jan. 9, 1995, which is a divisional of U.S. Ser. No. 07 / 874,344, now U.S. Pat. No. 5,407,816, filed Apr. 24, 1992, which is a continuation-in-part of U.S. Ser. No. 07 / 839,144, filed Feb. 20, 1992. The text of each priority application is expressly incorporated herein by reference to the extent that the text of the respective priority application differs from this application.BACKGROUND OF THE INVENTION [0002] A. Field of the Invention [0003] This invention is directed to methods for the enhanced production and recovery of taxol, baccatin III and other taxanes by cell cultures of Taxus species. [0004] B. Related Art The Taxane Supply Challenge [0005] Taxol is a diterpenoid alkaloid originally isolated from th...

AI Technical Summary

Benefits of technology

[0020] The objects of this invention include the formulation of special environmental conditions to foster rapid growth, high cell densities, and high cell viabilities. (The growth characteristics reported in this study surpass previous results by a significant factor.)

[0021] An object of this invention is to produce taxanes at high rates by careful selection of cell lines, careful choice and manipulation of medium conditions, incorporation of enhancement agents, and careful selection of process-operating modes.

[0023] The inventors have discovered that 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. Further, by the methods of this invention it is possible to obtain taxol, baccatin III, and other taxanes in a much shorter time frame than previously reported. In particular, the inventors found that the species, Taxus chinensis, is capable of rapid growth and of producing extremely high levels of taxol, baccatin III, and taxanes within a short period of time. With the species Taxus chinensis, the inventors have been able to manipulate cells to yield taxol, baccatin III, and taxanes in amounts far in excess of the amounts obtained from tissue cultures of the other Taxus species.

[0024] 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.

[0025] In one embodiment, this invention provides a method for producing taxanes in high yields in cell culture of a Taxus species comprising cultivating cells of a Taxus species in suspension culture in one or more nutrient media under growth and product formation conditions, and recovering one or more taxanes from said cells or said medium of said cell culture, or both, the cells being derived from callus or suspension cultures and the nutrient media containing an inhibitor of phenylpropanoid metabolism. Suitable inhibitors of phenylpropanoid metabolism include 3,4-methylenedioxy-6-nitrocinnamic acid, 3,4-methylenedioxycinnamic acid, 3,4-methylenedioxy-phenylpropionic acid, 3,4-methylenedioxyphenylacetic acid, 3,4-methylenedioxybenzoic acid, 3,4-trans-dimethoxycinnamic acid, 4-hydroxycinnamic acid, phenylpropiolic acid, fluorophenylalanine, 1-aminobenzotriazole, 2-hydroxy-4,6-dimethoxybenzoic acid, SKF-525A, ammonium oxalate, vinylimidazole, diethyldithiocarbamic acid, and sinapic acid.

Problems solved by technology

Total synthesis, while accomplished by academic laboratories, shows little promise as a viable commercial route to taxol.

Since aseptic, large-scale, plant cell cultivation is inherently expensive, a cell culture process becomes commercially relevant only when these costs are offset by high productivity.

A survey of secondary metabolite productivity among gymnosperm cultures also points to the difficulty of inducing rapid biosynthesis compared to herbaceous species.

However, these results were obtained with a slow-growing (30% biomass increase in 18 days) and low cell density (5 to 7 grams dry weight per liter) culture.

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