Genetic Instability of Productivity

A loss of productivity is a critical factor in industrial applications. The continuous maintenance of cultured cells will lead to the accumulation of mutations, as previously mentioned (Hirochika et al., 1996; Phillips et al., 1994). Currently, the only means to avoid the mutational loss of productivity is cryopreservation, although some empirical trials have shown that repeated selection can overcome this problem based on the establishment of cell lines that are more stable for production (Sato and Yamada, 1984). In fact, selected C. japonica cells showed the full chromosome number of 18, while other cell lines showed quite different numbers of chromosomes (Sato et al., unpublished data; Yamada and Mino, 1986). Additional studies are needed to determine how to control spontaneous mutation/ genetic and epigenetic modification during culture.

While the molecular investigation of the loss of biosynthetic activity is rather limited, the recovery of biosynthetic activity after the regeneration of whole plants is common as is a loss of productivity that accompanies a phenotype abnormality. Some secondary metabolites might be involved in the normal development of plants; for example, the cosuppression of PMT in tobacco induced an abnormal morphology (Sato et al., 2001). The silencing of obtusifoliol-14α-demethylase (CYP51) in sterol biosynthesis also reduced the growth of transgenic tobacco (Burger et al., 2003).

Fluctuation of biosynthetic activity is more frequently observed, especially during continuous subculture. For example, the accumulation of alkaloids by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors (Whitmer et al., 2003). High transgene-encoded TDC activity was not only unnecessary for increased productivity but also detrimental to the normal growth of the cultures (Canel et al., 1998). Since primary and secondary metabolism compete for the pool of substrates, it is quite likely that rapidly growing cells consume substrates so that none are available for production of secondary metabolites, for example, rapid growth but poor alkaloid production. The optimization of cell growth and metabolite production are important issues for industrialization. We point in particular to two-step culture methods that promote growth and metabolite production separately as essential to overcome these constraints (Fujita et al., 1986).

Another reason for fluctuation is the active channeling (catabolism) of metabolites. We sometimes encounter changes in metabolites within cells, which suggests that metabolism is not static. There is often a difference in the metabolite profile between intact tissue and cultured cells (Fu, 1998; Ketchum et al., 2003). A recent metabolite profile analysis of alkaloids in camptothecin-producing plants showed that anthraquinones, which can be considered phytoalexins, were present in the extracts of hairy roots and calli but not in the differentiated plant of O. pumila (Yamazaki et al., 2003). This is just one example of a difference in composition between differentiated plants and hairy roots callus tissues. The poor productivity of opium poppy cells for morphinan alkaloids might be due to the active channeling of intermediates into benzophenanthridine alkaloids rather than to morphinan alkaloids. Similarly, first attempts to reduce the pathway to benzophenanthridine alkaloid with antisense BBE gene in opium poppy plants have been unsuccessful or, at least, ambiguous (Frick et al., 2004). A detailed molecular characterization of metabolic flow should be helpful and clarify mechanisms that lead to the loss of productivity.

The long-term instability of alkaloid production is currently unavoidable, even when stable transformed cells are maintained in selective medium containing selective agents and GUS activity is monitored. For example, the activities of exogenous STR and TDC varied greatly over time, occasionally falling to the levels seen in nontransgenic cultures, indicating that indirect selection, such as antibiotic resistance, is insufficient to maintain the concerted expression of a secondary metabolite pathway necessary for high productivity (Whitmer et al., 2003).