cDNA cloning and functional characterization of squalene epoxidase in an oil plant. Euphorbia tirucalli

White latex in shoots of a subtropical shrub, Euphorbia tirucalli contains lots of secondary metabolites such as phytosterols and triterpenoids. Squalene epoxidase is a putative rate-limit enzyme in mevalonate pathway. In order to obtain a gene for this enzyme, we have performed RT-PCR cloning and obtained two cDNA sequences, which are completely identical in the open reading frame region, except two point mutations in the 244th and 296th codons from the N-terminal. Sequencing of genome DNA and Southern blot analyses indicated that the EtSE locus consisted of two alleles. An entire ORF sequence of the EtSE cDNA was cloned in pWV3 expression vector and introduced into an ERG1-disrupted yeast mutant, KLN1. Colonies of transformants with the EtSE, or wild-type ScERG1, grew in an aerobic condition on SD+Trp, or YPD media, which lack ergosterol. This work was partly performed as one of the technology development projects of the "Green Biotechnology Program" in New Energy and Industrial Technology Development Organization (NEDO).