While some metabolites can be produced in cell culture systems, or can be induced by signal mediators, the establishment of a high-yield culture, which is essential for commercialization, requires both high productivity and stability. In this regard, the starting materials should show suitable genetic diversity, especially since correlations between the productivity in the originator plant and in cultured cells have not been established (Suzuki et al., 1987). Thus, empirical trials are needed to establish cell lines with suitable metabolite productivity. For example, H. niger is a good plant species for producing tropane alkaloids in cell culture, whereas the productivity compared to the intact plant is lower in A. belladonna and D. stramonium cell cultures (Hashimoto et al., 1986).
The culture medium and culture conditions, including temperature, illumination, aeration, and so on, are also important factors. Other factors such as shearing stress, gas composition, and cell density are also important for expanding the scale of the culture (Bisaria and Panda, 1991; Matsubara et al., 1989; Taticek et al., 1994). For example, berberine production requires higher aeration because of the greater need for oxygen in the different biosynthetic steps (Sato and Yamada, 1984). Similarly, illumination clearly enhances terpenoid indole alkaloid production in Catharanthus cells (Kutchan et al., 1988). The optimization of culture conditions for production is not yet well understood; a more detailed characterization of the biosynthetic pathways and regulation mechanisms may help to guide such optimization.
Furthermore, the selection of high- and stable-metabolite-producing cells is crucial. Since plant cells change their ploidy condition during development and cell culture induces spontaneous mutation (Galbraith et al., 1991; Hirochika et al., 1996; Phillips et al., 1994), the selection of cells should be effective for establishing a high-betabolite-producing line (Sato and Yamada, 1984), while continuous cell culture also leads to instability (see below) (Deus-Neumann and Zenk, 1984).
The continuous maintenance of high- and stable-metabolite-producing cell lines is also important because subtle changes in the culture conditions and the transfer method can easily change the cell phenotype. One endogenous factor that influences productivity is the effect of metabolites that are produced on cell viability. Since many metabolites are biologically active, that is, cytotoxic, the high accumulation of these chemicals can induce cell death or growth retardation; for example, berberine inhibits the growth of nonberberine-producing plant cells (Sakai et al., 2002). The avoidance of cytotoxicity by the ectopic expression of an ABC transporter clearly indicated that metabolites themselves can be toxic if the cells lack a detoxification/segregation machinery or tolerance system (Goossens et al., 2003a). While the essential factors needed to stabilize a cell culture have not yet been identified, our experience suggests that slow-growing cells are more stable. This might be due to the presence of slow cell division and low mutation frequency, as well as less competition for primary metabolites between cell growth and secondary metabolism; a negative correlation is often observed between growth and alkaloid yield (Hashimoto et al., 1986). Alternatively, it seems appropriate to optimize the two-step growth and production system that has been employed for cell growth and shikonin production in Lithospermum erythrorizon (Fujita et al., 1986).
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