Humanizing yeast sterol biosynthesis to understand evolution and disease
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Baker’s yeast (S. cerevisiae) and humans (H. sapiens) have a relatively high level of genetic conservation in essential pathways including the proteasome, cytoskeleton, and sterol synthesis due to sharing a common ancestor approximately 1.1 billion years ago. This thesis focuses on the sterol biosynthesis pathway, which produces the related membrane lipids of ergosterol in yeast and cholesterol in humans, both of which are critical in maintaining cell membrane structure and fluidity. Yeast sterol biosynthesis genes are essential for yeast viability in standard lab growth conditions, however, previous studies in our laboratory have shown that yeast strains lacking genes for ergosterol biosynthesis can live if cholesterol is supplemented in the growth medium, suggesting these lipids may be interchangeable in yeast. To understand the extent to which yeast sterol biosynthesis can be humanized we systematically aimed to engineer this human pathway into yeast in place of its own genes. We took advantage of the CRISPR-Cas9 genetic engineering technique to systematically substitute yeast genes in the sterol pathway with their human equivalents. To date, we have humanized 80% of the yeast sterol biosynthesis pathway. Besides informing us about the evolution of core metabolic pathways across vast periods of divergence, a humanized yeast strain that contains the entire cholesterol biosynthesis pathway should serve as a remarkable new reagent for downstream medical research to help us better understand human lipid metabolic disease (including heart disease) and to perform high-throughput allelic variant and drug screens in this pathway.