BETA-AMYRIN SYNTHASE - CLONING OF OXIDOSQUALENE CYCLASE THAT CATALYZES THE FORMATION OF THE MOST POPULAR TRITERPENE AMONG HIGHER-PLANTS

beta-amyrin, a typical pentacyclic triterpene having an oleanane skele ton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)-2,3-oxidosqualene. The enzyme, beta-amyrin s ynthase, catalyzing the cyclization of oxidosqualene into beta-amyrin, generates five rings and eight asymmetric centers in a single transfo rmation. A homology-based PCR method was attempted to obtain the cDNA of this enzyme from the hairy root of Panax ginseng which produces ole anane saponins together with dammarane-type saponins. Two sets of dege nerate oligonucleotide primers were designed at the regions which are highly conserved among known oxidosqualene cyclases (OSCs). Nested PCR s using these primers successfully amplified the core fragment which r evealed the presence of two OSC clones PNX and PNY. Specific amplifica tion of each clone by 3'-RACE and 5'-RACE was carried out to obtain th e whole sequences. The two clones exhibited 60% amino acid identity to each other. A full-length clone of PNY was ligated into the yeast exp ression vector pYES2 under the GAL1 promoter to give pOSC(PNY). beta-a myrin production was observed with the mutant yeast lacking lanosterol synthase, transformed by this plasmid. The sequence of pOSC(PNY) cont ains an open reading frame of 2289 nucleotides which codes for 763 ami no acids with a predicted molecular mass of 88 kDa. Sequence compariso n with other OSCs showed a high level of similarity with lanosterol, c ycloartenol and lupeol synthases. The other clone, pOSC(PNX), was show n to be cycloartenol synthase by similar expression in yeast. The pres ent studies have revealed that distinct OSC exists for triterpene form ation in higher plants, and the high level of similarity with cycloart enol synthase indicates close evolutional relationship between sterol and triterpene biosynthesis.