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
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).