Enhancement of shikonin production in single- and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound

This work demonstrates the use of low-energy ultrasound (US) to enhance secondary metabolite production in plant cell cultures. Suspension culture of Lithospermum erythrorhizon cells was exposed to low-power US (power density < or = 113.9 mW/cm(3)) for short periods (1-8 min). The US exposure significantly stimulated the shikonin biosynthesis of the cells, and at certain US doses, increased the volumetric shikonin yield by about 60%-70%. Meanwhile, the shikonin excreted from the cells was increased from 20% to 65%-70%, due partially to an increase in the cell membrane permeability by sonication. With combined use of US treatment and in situ product extraction by an organic solvent, or the two-phase culture, the volumetric shikonin yield was increased more than two- to threefold. Increasing in the number of US exposures during the culture process usually resulted in negative effects on shikonin yield but slight stimulation of shikonin excretion. US at relatively high energy levels caused slight cell growth depression (maximum 9% decrease in dry cell weight). Two key enzymes for the secondary metabolite biosynthesis of cells, phenylalanine ammonia lyase and p-hydroxybenzoic acid geranyltransferase, were found to be stimulated by the US. The US stimulation of secondary metabolite biosynthesis was attributed to the metabolic activity of cells activated by US, and more specifically, the defense responses of plant cells to the mechanical stress of US irradiation.