A yeast with muscle does not run faster- full humanization of the glycolytic pathway in Saccharomyces cerevisiae

Humanization of single genes in yeast is a widely used strategy to explore gene functionality and study metazoan cellular processes, including those underlying human pathology. Recent synthetic biology advances can overcome the challenges posed by the high genetic redundancy of eukaryotic genomes and allow humanization of full pathways and processes. In this study we demonstrate full humanization of the key pathway of glycolysis. Through single gene complementation, full pathway humanization and laboratory evolution the functionality of 25 human glycolytic enzymes in yeast was explored. All enzymes except the hexokinases 1-3 were able to complement the catalytic function of their yeast orthologs and aldolase and enolase also complemented moonlighting functions outside glycolysis. Comparison with skeletal muscle cells showed that for most enzymes, transplantation in yeast did not affect their turnover number. This central metabolic pathway was therefore highly functional in yeast, opening the door to promising models for metazoans.