ISOLATION OF AN ARABIDOPSIS-THALIANA GENE ENCODING CYCLOARTENOL SYNTHASE BY FUNCTIONAL EXPRESSION IN A YEAST MUTANT LACKING LANOSTEROL SYNTHASE BY THE USE OF A CHROMATOGRAPHIC SCREEN

Whereas vertebrates and fungi synthesize sterols from epoxysqualene th rough the intermediate lanosterol, plants cyclize epoxysqualene to cyc loartenol as the initial sterol. We report the cloning and characteriz ation of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synt hase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epox ysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was tra nsformed with an A. thaliana cDNA yeast expression library, and coloni es were assayed for epoxy-squalene mutase activity by thin-layer chrom atography. One out of almost-equal-to 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloar tenol. This activity was shown to be plasmid dependent. The plasmid in sert contains a 2277-bp open reading frame capable of encoding an 86-k Da protein with significant homology to lanosterol synthase from Candi da albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus ac idocalcarius. The method used to clone this gene should be generally a pplicable to genes responsible for secondary metabolite biosynthesis.