A yeast with muscle doesn’t run faster: full humanization of the glycolytic pathway in Saccharomyces cerevisiae

Summary While transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper the humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of both glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light novel, context-dependent responses. Remarkably, human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, while hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed the preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire, essential pathway paves the way to the establishment of species, tissue and disease-specific metazoan models. One Sentence Summary This work demonstrates the successful humanization of an entire pathway in Saccharomyces cerevisiae and establishes an attractive strategy to study (human) glycolysis architecture and regulation. Highlights The successful humanization of the entire glycolytic pathway in yeast offers new microbial models for both fundamental and applied studies. Both glycolytic and moonlighting functions and turnover numbers of glycolytic enzymes are highly conserved between yeast and human. Functionality of human hexokinases 1 and 2 in yeast requires mutations in the catalytic or allosteric binding sites. Combination of single gene and full transplantation with laboratory evolution reveals context-dependent activity and evolution of glycolytic enzymes.