In situ extraction of benzophenanthridine alkaloids from California poppy suspension cultures

Plant cell culture is an established alternative to the harvest and extraction of whole plant material for the production of valuable secondary metabolites. There are a number of drawbacks to plant cell culture that have limited its use on a large industrial scale, including the low yield of metabolite products and the retention of products within the cells. Techniques, including elicitation and in situ product extraction, have been developed to overcome these limitations and successfully increase product yields. The Eschscholzia californica (California poppy) plant cell culture system has been successfully treated with both techniques to increase yields of its benzophenanthridine alkaloid products. The application of in situ extraction using the triglyceride tricaprylin as an extractant phase is particularly interesting as the alkaloid profiles depend on more than the passive accumulation of extracellular product.; This work shows that the tricaprylin second phase results in a rapid 8-fold increase in alkaloid productivity with a response time similar to that of elicited cultures, although there is no corresponding increase in intracellular or extracellular media alkaloid product levels. Additionally, the application of in situ extraction does not induce ethylene synthesis, an essential indicator of elicited metabolic activation. This suggests that accumulation of the metabolite products from the media and relief of feedback inhibition based on limited intracellular or extracellular storage space is an important factor that promotes further synthesis under two-phase conditions.; The California poppy system is also shown to be a highly vacuolated cell line and these vacuoles appear to be the final storage site of the benzophenanthridine alkaloids. Transport of the alkaloids in and out of these vacuoles is found to occur by simple diffusion with first-order kinetics and not by an active transport process.; Using this transport data as well as data on the growth and substrate utilization in the system, a model was developed to describe the biomass synthesis and alkaloid productivity of the cultured cells. This model incorporates the effects of elicitation and in situ product extraction and is seen to effectively simulate the experimentally observed productivity enhancement. The model also suggests that some metabolic activation, at least partially independent of the elicitation signaling process, must be induced by the second phase and that both accumulation of alkaloid product and activation of metabolism are responsible for the dramatic yields observed with the application of in situ extraction.