Isolation and characterization of an oxidosqualene cyclase gene encoding a β-amyrin synthase involved in Polygala tenuifolia Willd. saponin biosynthesis

Abstract

Polygala tenuifolia Willdenow is a rich source of triterpene saponins, onjisaponins and polygalasaponins, used as herbal medicine to treat phlegms and for detumescence in traditional Asian healing. The Polygala saponins share the oleanane backbone structure and are, therefore, likely synthesized via β-amyrin as a common precursor. We hypothesized that, in analogy to diverse other plant species, this central intermediate should be formed by a β-amyrin synthase catalyzing the complex cyclization of oxidosqualene. This member of the oxidosqualene cyclase (OSC) family of enzymes is thus defining an important branch point between primary and secondary metabolisms, and playing a crucial role in the control of oleanane-type triterpene saponin biosynthesis. From P. tenuifolia roots, we isolated an OSC cDNA containing a reading frame of 2,289 bp nucleotides. The predicted protein of 763 amino acids (molecular weight 87.353 kDa) showed particularly high amino acid sequence identities to known β-amyrin synthases (85-87 %) and was, therefore, named PtBS. Expression of PtBS in the triterpenoid synthase-deficient yeast mutant GIL77 led to the production of β-amyrin as sole product. qRT-PCR analysis of various P. tenuifolia organs showed that PtBS transcript levels were highest in the roots, consistent with onjisaponin accumulation patterns. Therefore, we conclude that PtBS is the β-amyrin synthase enzyme catalyzing the first committed step in the biosynthesis of onjisaponins and polygalasaponins in P. tenuifolia.

Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd

“…In our datasets, eleven unigenes for OSC were determined; and three bAS and eight CAS unigenes were subsequently annotated. Out of those, the sequences of three unigenes for bAS were consistent with those of the PtBS gene (EF107623) reported by Jin et al [ 15 ]. Eight unigenes for CAS appeared to have open reading frames (ORFs) while two unigenes possessed full-length cDNAs that were identified by comparing amino acid sequences.…”

Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii

“…The recombinant protein expressed from SgCAS-pET28a was mostly insoluble in the prokaryotic system; however, yeast can provide the substrate naturally. Thus, we can introduce SgCAS into yeast to identify its function like other OSCs24, 25, 26, 27, 28. SgCAS was also highly expressed in fruits with an expression pattern similar to that of cucurbitadienol synthase (data not published), indicating SgCAS was a competitor for the substrate 2,3-oxidosqualene with SgCAS.…”

Cloning and analysis of β-amyrin synthase gene in Bupleurum chinense

“…(Liu et al 2009), AsOXA1 from Aster sedifolius L. (Cammareri et al 2008), GgbAS1 from Glycyrrhiza glabra L. (Hayashi et al 2001), PNY from Panax ginseng C. A. Mey (Kushiro et al 1998), LjOSC1 from Lotus japonicas L. (Sawai et al 2006), and PtBS from Polygala tenuifolia Willd. (Jin et al 2014). Besides, a few of multifunctional OSCs, which produce multiproduct such as CSOSC2 producing lupeol, germanicol, and b-amyrin in Costus speciosus (Koen.)…”

“…1 amyrin, a-amyrin or lupeol, with oleanane, ursane or lupane ring structures, respectively. OSCs, therefore, control the biosynthesis of various triterpenoid ring structures, and the characteristic mixture of triterpenoid backbones in each plant species is determined by its set of OSCs and their product profiles (Jin et al 2014). The cDNAs of b-AS have been cloned and functionally characterized in many plant species including GsAS1 from Gentiana straminea Maxim.…”