Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment.

Abstract

The main objective of the study is molecular and biological characterization of the human-yeast hybrid squalene synthase (SQS), as a promising target for treatment of hypercholesterolaemia. The human-yeast hybrid SQS, with 67% amino acids, including the catalytic site derived from human enzyme, was expressed in Saccharomyces cerevisiae strain deleted of its own SQS gene. The constructed strain has a decreased level of sterols compared to the control strain. The mevalonate pathway and sterol biosynthesis genes are induced and the level of triacylglycerols is increased. Treatment of the strain with rosuvastatin or zaragozic acid, two mevalonate pathway inhibitors, decreased the amounts of squalene, lanosterol and ergosterol, and up-regulated expression of several genes encoding enzymes responsible for biosynthesis of ergosterol precursors. Conversely, expression of the majority genes implicated in the biosynthesis of other mevalonate pathway end products, ubiquinone and dolichol, was down-regulated. The S.cerevisiae strain constructed in this study enables to investigate the physiological and molecular effects of inhibitors on cell functioning. The yeast strain expressing hybrid SQS with the catalytic core of human enzyme is a convenient tool for efficient screening for novel inhibitors of cholesterol-lowering properties.