Full humanization of the glycolytic pathway in Saccharomyces cerevisiae
Francine Boonekamp (TU Delft - BT/Industriele Microbiologie)
Ewout Knibbe (TU Delft - BT/Industriele Microbiologie)
Marcel A. Vieira Lara (University Medical Center Groningen)
M. Wijsman (TU Delft - BT/Industriele Microbiologie)
MAH Luttik (TU Delft - BT/Industriele Microbiologie)
Karen van Eunen (University Medical Center Groningen)
M.J. Ridder (TU Delft - BT/Industriele Microbiologie)
Martin Pabst (TU Delft - BT/Environmental Biotechnology)
Jean Marc G. Daran (TU Delft - BT/Industriele Microbiologie)
P.A.S. Lapujade (TU Delft - BT/Industriele Microbiologie)
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Abstract
Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper 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 glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed 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 for establishment of species-, tissue-, and disease-specific metazoan models.
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