Methyl jasmonate as a control factor of the synthase squalene gene promoter and ginsenoside production in American ginseng hairy root cultured in shake flasks and a nutrient sprinkle bioreactor

Abstract

Hairy root cultures of Panax quinquefolium (L.) produce triterpenoid saponins: ginsenosides with broad medical applications. The crucial enzyme in the process of ginsenoside biosynthesis is squalene synthase (SSq). In this study a 741 bp fragment of the P. quinquefolium SSq gene, consisting of a proximal promoter, 5′UTR (5′ untranslated region) and 5′ CDS (coding DNA sequence) was isolated. An analysis of an isolated fragment with in silico tools indicated a lack of tandem repeats, miRNA binding sites and CpG/CpNpG elements. However, the proximal promoter contained potential cis-elements, mediating the response to multiple external stimuli, including light, heat-stress and drought. Among them, two CGTCA motifs could be involved in the response to methyl jasmonate (MeJA) treatment. To evaluate the functional significance of MeJA on P. quinquefolium SSq expression, quantitative RT-PCR experiments were performed at different elicitor concentration. Additionally, the effect of methyl jasmonate on ginsenoside biosynthesis was examined at 5, 50, 100, 250, 500 μM l−1 concentrations. Experiments were performed in shake flasks and a nutrient sprinkle bioreactor offering better oxygenation rate and potential for future scaling-up of the biosynthesis process. The saponin content was determined using HPLC. In shake flask and bioreactor culture, the maximum yield (respectively 27.33 mg g−1 d.w. and 51.0 mg g−1 d.w.) of the sum of six examined ginsenosides was achieved in modified Gamborg B-5 medium containing 250 μM l−1methyl jasmonate after seven days of elicitation. Rb1 (20(S)protopanaxadiol-3-[O-β-d-glucopyranosyl(l → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranosyl(1 → 6)-β-d-glucopyranoside) and Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(l → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) metabolites quantitatively dominated both in shake flask and bioreactor cultures. The level of Rb1 increased 5.6-fold and achieved 14.35 mg g−1 d.w. of hairy roots cultivated in flasks. In bioreactor cultures, this metabolite achieved 24.77 mg g−1 d.w. increasing its content 2.6-fold compared to control. The level of Re increased 1.8-fold and reached 4.92 mg g−1 d.w. in flask cultures. In bioreactor cultures the content of this metabolite was 6.23 mg g−1 d.w. The highest productivity of ginseng saponins (15.37 mg g−1d.w. l−1 day−1) was noted in bioreactor hairy root cultures after 7 days elicitation of 250 μM l−1 MeJA.