Improved anthraquinone accumulation in cell cultures of Cinchona `Robusta' by feeding of biosynthetic precursors and inhibitors

The effects of feeding of biosynthetic precursors and pathway specific inhibitors on anthraquinone (AQ) accumulation in fungal elicited cell cultures of Cinchona `Robusta' were studied. Addition of glyceraldehyde (1 mM), the initial precursor in the methyl-d-erythritol 4-phosphate (MEP) pathway, did not increase AQ accumulation, suggesting that the endogenous level of this precursor is not a limiting factor of AQ flux. It is proposed that AQs in Cinchona might be derived from the phenylpropanoid pathway, e.g. from caffeic acid. Addition of ferulic acid (1 mM) did not stimulate AQ accumulation, while addition of caffeic acid increased AQ accumulation by 48% compared to the control. The stimulating effect of feeding caffeic acid on AQ accumulation might be due to activation of other pathways. Addition of tectoquinone (2-methyl-anthraquinone) did not change the AQ patterns nor the shifts between AQs in control and tectoquinone-treated cell cultures. Addition of lovastatin, a specific inhibitor of the mevalonic acid pathway, did not inhibit the AQ accumulation. Clomazone, an inhibitor in the MEP pathway, inhibited the AQ accumulation, however. The simultaneous addition of lovastatin and clomazone inhibited both cell growth and AQ accumulation. These results further support the finding that isopentenyl diphosphate, which constitutes ring C of AQs in Cinchona `Robusta', is derived from the MEP pathway, and not from the mevalonic acid pathway.