Cloning and Functional Analysis of a &beta;-Amyrin Synthase Gene Associated with Oleanolic Acid Biosynthesis in <i>Gentiana straminea</i> M<small>AXIM.</small>

Abstract

Gentiana straminea MAXIM. (Mahuaqinjiao) belongs to theGentianaceae family, and is an important medicinal plant in China. It is found on the Qinghai-Tibetan plateau at altitudes between 2000 and 5000 m asl, 1) and is a rich source of secoiridoid glucosides, including gentiopicrosidel, loganic acid, swertiamarin, oleanolic acid and roburic acid, 2) all of which have been associated with medicinal properties. The triterpenoid oleanolic acid has been used to treat hepatitis and a number of diseases of the immune system. 3,4) Wild populations of G. straminea have been threatened by excessive excavation, and its seeds germinate poorly in low altitude environments.5) Hence there is a need to enhance the production of oleanolic acid by secondary metabolic engineering in the condition of raw G. straminea limited.b-amyrin is thought to act as a precursor for oleanolic acid, and is associated with oleanolic acid production.6) It is synthesized within the isoprenoid pathway by the cyclization of 2,3-oxidosqualene, catalysed by b-amyrin synthase (an oxidosqualene cyclase, or OSC) (Fig. 1). A number of OSCs have been cloned to date, and their enzyme functions identified by heterologous expression in yeast. 7,8) These enzymes can have either mono-or multifunctional activity in higher plants, and yield various triterpene products.9-11) Furthermore, some research shows that OSCs are key enzymes in the isoprenoid pathway to form different products.12-16) Thus b-amyrin synthase in G. straminea may represent a key enzyme for oleanolic acid synthesis, and the cloning of its encoding gene is required for any effort to raise the content of a secondary metabolite such as oleanolic acid using metabolic engineering.Methyl jasmonate (MeJA) is an elicitor which enhances the content of triterpene saponins. [17][18][19][20] The indications are that the accumulation of triterpene saponins is related to OSC over-expression, mediated by MeJA. Thus, the relationship between OSC gene expression and oleanolic acid production in the presence of MeJA may be critical to our understanding of the mechanism of secondary product accumulation. Here, we describe the characteristics of G. straminea bamyrin synthase, and show how manipulation of its expression level affects the quantity of oleanolic acid in the presence of MeJA. MATERIALS AND METHODSPlant Materials G. straminea samples were collected from Yushu Country, Qinghai Province, China. Voucher specimens have been deposited in Qinghai Normal University.Bacterial and Yeast Strains and Vectors Escherichia coli strain BL21(DE3) and the pET28(a) vector provided a prokayotic expression system. Bacterial cells were grown at 37°C in LB medium containing 0.5% (w/v) yeast extract, 1% (w/v) tryptone, 1% (w/v) NaCl and 100mg/ml kanamycin. Pichia pastoris strain X-33 and the pPICZA vector provided an eukaryotic expression system. Yeast cells were grown at 30°C in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose supplemented with 100 mg/ml Zeocin. Media were solidified with 2% ...

Molecular characterization and differential expression studies of an oxidosqualene cyclase (OSC) gene of Brahmi (Bacopa monniera)

“…In contrast, the expression level was highest in flower, and roots and hypocotyls in case of Nigella sativa (Scholz et al 2009) and Glycyrrhiza glabra (Hayashi et al 2001) respectively. In other reports, the accumulation of transcripts was higher in leaves of Gentiana straminea (Liu et al 2009) and C. asiatica (Kim et al 2005). …”

β‐Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae

“…Another report described by Liu et al . 38 also indicated that MeJA treatment upregulated the Gentiana straminea βAS expression levels and oleanolic acid accumulations. Conclusively, MeJA treatment can stimulate the accumulation of oleanane‐type saponins or sapogenins and the expression level of βAS gene.…”

Cloning and Functional Analysis of Geraniol 10-Hydroxylase, a Cytochrome P450 fromSwertia mussotiiFranch

“…Expression of SmG10H in Pichia pastoris yeast was carried out using an EasySelect Pichia Expression Kit (Invitrogen) following Liu et al 20) The membrane fractions, containing SmG10H recombinant protein, were isolated by differential centrifugation between 200 and 20;000 Â g as described by Collu et al…”