Abstract

Baker's yeast, or Saccharomyces cerevisiae, shares thousands of genes with humans, making it invaluable for studying human gene function, genetic interactions, and evolution of critical cellular processes. Humanized yeast, i.e., yeast with human genes functionally replacing their yeast orthologs, enables the direct study of human gene function and their disease-causing variants in a simplified model. Recent systematic functional complementation assays performed one gene at a time revealed that genes belonging to pathways or complexes are either entirely replaceable or are not. In the case of genes that are not easily replaceable, we hypothesize that some essential interactions have evolved in a species-specific manner, resulting in incompatibility. Therefore, humanizing whole processes may reveal incompatibilities towards humanization, by restoring local genetic or physical interactions, allowing replaceability of those genes. We tested this approach to humanizing the sterol biosynthesis pathway in its entirety in yeast. Using marker-less CRISPR-Cas9 selection and Homology Directed Repair, we show that several human genes can replace their yeast equivalents at their native loci generating single-gene humanized strains. Next, we demonstrate that multiple yeast genes belonging to the sterol biosynthesis pathway are replaceable in a single strain using a sequential approach (10 of 16 genes). Characterization of the humanized sterol strains reveals the impact of humanization on the fitness of yeast such as altered growth rates and temperature sensitivity, and proteome-scale changes affecting other biosynthesis pathways. Finally, we design a novel strategy to increase the efficiency of functional replaceability to provide a clear readout of replaceability. In future, these strategies will enable building an entirely human sterol biosynthesis pathway in yeast.