Long-term bioreactor cultivation affects dioscin content and the ratio of 25(S)- and 25(R)-protodioscin isomers in the suspension cell culture of Dioscorea deltoidea Wall.

Bioreactor-grown plant cells have emerged as a sustainable, high-quality source of plant biomass and bioactive phytochemicals alternative to overcollection of pharmaceutically important wild plant species. At the same time, concerns were raised about the potential biosynthetic instability of plant cell cultures during long-term bioreactor cultivation, which was rarely investigated. In this work, this concern was addressed by performing the first long-term (1.5 years) uninterrupted cultivation of Dioscorea deltoidea cell suspension in a 20-L bubble-type bioreactor using fill-and-draw mode with simultaneous monitoring of major bioactive compounds - steroidal glycosides protodioscin and dioscin, using HPLC-ESI-MS. In addition, the ratio of 25(S)/25(R)-isomers of protodioscin showing different pharmacological activities was monitored during the entire cultivation period. The results demonstrated that cell culture productivity (0.33 g/(L·day)), maximum dry weight accumulation (8.5 g/L), viability (80.2 %), and the total content of steroidal glycosides (1.74 % of dry weight) remained high during the entire cultivation. However, the content of dioscin, a spirostanol steroidal glycoside, decreased by 82 % after one year of cultivation. Moreover, the ratio of 25(S)/25(R)-isomers of protodioscin, a furostanol steroidal glycoside, in the cell biomass changed reversely from 0.66 to 1.40 after the first half-year of the cultivation. These results evidenced the complex dynamics of steroidal glycosides biosynthesis in plant cell cultures during the prolonged bioreactor cultivation and advocate for the importance of monitoring both the concentration and the isomeric composition of the desired metabolites to assure high quality of the biotechnologically produced cell biomass.