Transgenic hairy roots: recent trends and applications

Pharmaceutically significant secondary metabolites or phytopharmaceuticals include alkaloids, glycosides, flavonoids, volatile oils, tannins, resins etc. Currently, most of these secondary metabolites are isolated from wild or cultivated plants because their chemical synthesis is either extremely difficult or economically infeasible. Biotechnological production in plant cell cultures is an attractive alternative, but to date this has had only limited commercial success because of a lack of understanding of how these metabolites are synthesized. Plants and/or plant cells in vitro, show physiological and morphological responses to microbial, physical or chemical factors which are known as ‘elicitors’. Elicitation is a process of induced or enhanced synthesis of secondary metabolites by the plants to ensure their survival, persistence and competitiveness. Here, we discuss the classification of elicitors, their mechanism of action, and applications for the production of phyto-pharmaceuticals from medicinal plants.

Plant cell elicitation for production of secondary metabolites: A review

Plants are the tremendous source for the discovery of new products with medicinal importance in drug development. Today several distinct chemicals derived from plants are important drugs, which are currently used in one or more countries in the world. Secondary metabolites are economically important as drugs, flavor and fragrances, dye and pigments, pesticides, and food additives. Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Plant cell and tissue culture technologies can be established routinely under sterile conditions from explants, such as plant leaves, stems, roots, and meristems for both the ways for multiplication and extraction of secondary metabolites. In vitro production of secondary metabolite in plant cell suspension cultures has been reported from various medicinal plants, and bioreactors are the key step for their commercial production. Based on this lime light, the present review is aimed to cover phytotherapeutic application and recent advancement for the production of some important plant pharmaceuticals.

Current approaches toward production of secondary plant metabolites.

Taxus cell culture may be an alternative source of paclitaxel and related taxane production Significantly increased amounts of paclitaxel and baccatin III were observed in cultured cells of Taxus species after exposure to methyl jasmonate Among the three species of Taxus tested, Taxus media showed the highest paclitaxel content while Taxus baccata showed the highest baccatin III content when 100 microM of methyl jasmonate was added to the culture media Furthermore, the activities of methyl jasmonate and related substances for inducing paclitaxel production were compared in cell suspension cultures of T media Methyl jasmonate and its free acid showed the strongest promoting activity Reduction of the keto group at the C-3 position greatly reduced this activity cis-Jasmone, which does not have a carboxyl group at the C-1 position, had almost no activity These results suggest that these two regions of methyl jasmonate are important for promoting the production of paclitaxel and related taxanes in Taxus cell cultures

Methyl jasmonate-induced overproduction of paclitaxel and baccatin III in Taxus cell suspension cultures.

Terpenoids are a diverse class of natural products that have many functions in the plant kingdom and in human health and nutrition. Their chemical diversity has led to the discovery of over 40,000 different structures, with several classes serving as important pharmaceutical agents, including the anticancer agents paclitaxel (Taxol) and terpenoid-derived indole alkaloids. Many terpenoid compounds are found in low yield from natural sources, so plant cell cultures have been investigated as an alternate production strategy. Metabolic engineering of whole plants and plant cell cultures is an effective tool to both increase terpenoid yield and alter terpenoid distribution for desired properties such as enhanced flavor, fragrance or color. Recent advances in defining terpenoid metabolic pathways, particularly in secondary metabolism, enhanced knowledge concerning regulation of terpenoid accumulation, and application of emerging plant systems biology approaches, have enabled metabolic engineering of terpenoid production. This paper reviews the current state of knowledge of terpenoid metabolism, with a special focus on production of important pharmaceutically active secondary metabolic terpenoids in plant cell cultures. Strategies for defining pathways and uncovering rate-influencing steps in global metabolism, and applying this information for successful terpenoid metabolic engineering, are emphasized.

Production and engineering of terpenoids in plant cell culture

The kinetics of biomass accumulation, nutrient uptake and taxol production of Taxus baccata cell suspensions were examined in three bioreactor configurations, viz. 250-mL Erienmeyerflasks, 1-L working volume pneumatically mixed (PMB), and stirred tank (STB) bioreactors. Qualitatively similar kinetics were observed in all three bioreactor types. Biomass accumulation and specific nutrient uptake rates exhibited biphasic characteristics. Carbohydrate uptake and biomass accumulation substantially ceased when phosphate was depleted from the medium. Phosphate was identified as a possible growth-limiting nutrient. Taxol accumulated exclusively in the second phase of growth. A maximum taxol concentration of 1.5 mg/L was obtained in the PMB which was fivefold greater than that obtained in the Erienmeyer flasks and the STB, but the relative kinetics of taxol production was the same in all three reactor types. Biomass yields were calculated from the kinetic data and a stoichiometry for biomass formation was evaluated. The similarity of kinetics in the three bioreactor configurations suggests that taxol production by T. baccata cell suspensions is amenable to scateup. (c) 1995 John Wiley & Sons, Inc.

Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata

The addition of cell extracts and cultures filtrate of Pencillium minioluteum, Botrytis cinerea, Verticillium dahliae, and Gilocladium deliqucescens on the tenth day after transferring Taxus sp. (RO1-M28) cell suspensions into an induction medium, further improved the production of Taxol
 Taxol is a registered trademark of Bristol Myers Squibb for paclitaxel.
 and total taxanes. Arachidonic acid (1mg/L) addition at the time of inoculation increased Taxol production by 150%. Oxidative stress induction and copper sulphate or sodium orthovanadate addition had no effect on Taxol production. Three categories of elicitors; those specifically stimulating Taxol production, those specifically stimulating the producion of other taxanes, and those stimulating taxane production uniformly, could be identified. The biosynthetic site of action of these elicitors is currently not known.

Elicitation of Taxus sp. cell cultures for production of taxol

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1−1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1−1) and low (<0.1 mg 1−1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1−1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1−1.

Taxol production in suspension cultures of Taxus baccata

Inhibition of biosynthetic enzymes and translation and translocation processes, elicitation, and precursor feeding were used to probe biosynthetic pathway compartmentation, substrate-product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1-95) The results suggest the following : (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precusor of Taxol (Taxol is a registered trademark of Bristol-Myers Squibb for paclitaxel); (iii) baccatin III appears to have cytoplasmic and plastidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol production but does not have a significant effect on baccatin III yield Semiempirical mathematical models were used to describe these results and predict potential yield-limiting steps Model simulations suggest that, under current operating conditions, Taxol production in Taxus chinensis (PRO1-95) cultures is limited by the ability of the cells to convert phenylalanine to phenylisoserine rather than by the branch-point acyl transferase This result is supported by the lack of improvement of Taxol yield by feeding phenylalanine or benzoylglycine The methods described in this article, while specifically expanding our knowledge of taxane production in PRO1-95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in plant cell cultures, especially when details of the pathway and compartmentation are sparse

Venkatesh Srinivasan

Papers

Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata

Elicitation of Taxus sp. cell cultures for production of taxol

Taxol production in suspension cultures of Taxus baccata

Metabolic inhibitors, elicitors, and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures.