Cloning, Characterization and Bacterial Expression of the Β-amyrin Synthase Gene from Panax Japonicus C. A. Mey

Panax japonicus C. A. Mey. is one of the rare traditional Chinese herbal medicines; its main active ingredient is ginsenosides. β-amyrin synthase (βAS) is the rate-limiting enzyme in the biosynthesis pathway of oleanane-type ginsenosides. We cloned for the first time the full-length cDNA of βAS from P. japonicus (GenBank: KP658156) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and we named it PjβAS, which contained an open reading frame (ORF) of 2,286 bp in length and encoded a protein of 761-amino acids in length and about 87.8 kDa in molecular weight. Bioinformatics analysis showed that PjβAS had more than 80% homology with βAS from P. ginseng, Aralia elata, Betula platyphylla, and Malusx domestica, etc. PjβAS consists of six functional domains of the terpene synthase family and the functional domain of the prenyltransferase/squalene oxidase family. The coding sequence of PjβAS was cloned into a prokaryotic expression vector pET-41a(+); and the recombinant vector pET-AS was transformed into the Escherichia coli BL21(DE3) strain. Real-time fluorescence quantitative PCR (qRT-PCR) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that PjβAS could be over-expressed by IPTG-induction in BL21(DE3). High performance liquid chromatography (HPLC) analysis showed that pET-AS could synthesize β-amyrin in BL21 (DE3) cells. These results indicated that PjβAS functions as a βAS gene. Introduction Panax japonicus C.A. Mey., belonging to Araliaceae Panax, is one of the rare traditional Chinese herbal medicines. All plants of the Panax genus have high medicinal value and their main active ingredient is ginseng saponins (Yun 2001). The total saponin content of the roots of P. ginseng is 2 7%, whereas the total saponin content in the roots of P. japonicus can be as high as 15%, which is 2 to seven-fold higher than that observed in P. ginseng and three-fold higher than reported in P. quinquefolius. Ginsenosides are further classified into the oleanane type and the dammarane type; and the oleanane-type saponins are the main saponins in P. japonicus (Haralampidis et al. 2002, Lichtenthaler et al. 1997). Ginsenosides are triterpenoid saponins of plant secondary metabolites, i.e. the product of the triterpenoid saponin biosynthesis branch in the isoprenoid pathway (Fig. 1). Triterpenoid saponins are formed by different cyclizations of squalene (Haralampidis et al. 2002; Lichtenthaler et al. 1997). There are two pathways for the synthesis of isopentenyl pyrophosphate (IPP) in the isoprenoid pathway, one is mevalonate (MVA) pathway (Chappell 1995, Goldstein and Brown 1990), and the other is the 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway, also known as the non-mevalonate pathway (Lichtenthaler 1999, Wanke et al. 2001). IPP in the triterpenoid biosynthesis pathway comes from the MVA pathway, and IPP can further synthesize squalene. *Author for correspondence: . College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China.