Enhanced anthraquinone biosynthesis in Damnacanthus major cells via medium optimization and fed-batch bioreactor culture.

Damnacanthus major is biotechnologically valued for its rich anthraquinone metabolites. However, limited wild resources necessitate the development of efficient cell culture production. This study first used single-factor experiments to determine the favorable concentrations of nitrogen, phosphorus, and calcium in the Murashige and Skoog (MS) medium. Building upon these findings, a three-factor, three-level orthogonal design was implemented to optimize the macronutrient composition. The optimal medium consisting of 90 mM nitrate, 1.25 mM phosphate, and 3.0 mM calcium and significantly increased dry biomass (12.22 g/L) and anthraquinone content (7.11 mg/g DW). Subsequently, a fed-batch bioreactor culture was conducted to evaluate the effects of different feeding frequencies. The three-feeding-strategy (FBC-3) produced the highest biomass (498.94 g/L fresh; 22.57 g/L dry), anthraquinone content (5.47 mg/g DW), and productivity (123.57 mg/L), representing improvements of 1.6- to 3.0-fold over batch culture. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis revealed the presence of seven anthraquinones, with rubiadin and rhein emerging as the predominant compounds. This study establishes an efficient, scalable system for producing anthraquinones from D. major cells, supporting the industrial application of plant cell-derived metabolites.