Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) ami no acid sequence of Saccharomyces cerevisiae identified an EST clone corres ponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This e nzyme catalyses the synthesis of isopentenyl diphosphate, the building bloc k of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predict ed amino acid sequence presents about 55% identity with the yeast, human an d rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then clo ned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 s train deficient in MVD, and the lethal phenotype of an ERG19 deleted strain . However, the wild-type sterol content was not fully restored suggesting t hat the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enz ymes.