Oxidosqualene cyclases from cell suspension cultures of Betula platyphylla var. japonica: molecular evolution of oxidosqualene cyclases in higher plants.

Betula platyphylla var. japonica is a rich source of triterpenoid as it contains dammarane type triterpenes in the leaves, and lupane type and oleanane type triterpenes in the bark. Four oxidosqualene cyclase cDNAs (BPX, BPX2, BPW and BPY) were cloned by homology based PCR methods from cell suspension cultures of B. platyphylla var. japonica. Open reading frames consisting of 2274, 2304, 2268 and 2340 bp were ligated into yeast expression plasmid pYES2 under the control of GAL1 promoter and introduced into lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and/or accumulated products in the transformants demonstrated that they encode cycloartenol synthase (BPX and BPX2), lupeol synthase (BPW) and beta-amyrin synthase (BPY) proteins. Phylogenetic tree was constructed for all the known oxidosqualene cyclases (OSCs) including the clones obtained in this study, revealing that OSCs having the same enzyme function form respective branches in the tree even though they derive from different plant species. Intriguing correlation was found between reaction mechanism and molecular evolution of OSCs in higher plants.