Supplementary Material pertaining to the manuscript:
What’s wrong with SwYG? Reaching the limits of S. cerevisiae molecular and analytical
Ewout Knibbe, Eline D. Postma, Francine
J. Boonekamp, Sofiia Dashko, Jordi Geelhoed,
Anne-Marijn Maat, Marijke A.H. Luttik, Marcel van den Broek,
construction of powerful cell factories requires extensive remodelling of
microbial genomes, entailing many rounds of transformations to perform the
large number of desired gene modifications. However, increasing the number of
genetic interventions inevitably increases the occurrence of unwanted mutations
and effects. Using glycolysis as paradigm, a previous study developed a Saccharomyces cerevisiae strain in which
the glycolytic genes, relocated to a single locus, can be easily swapped by any
new design, thereby enabling fast and easy remodelling of the entire pathway.
After 27 genetic modifications performed in 43 transformation rounds, the
Switchable Yeast Glycolysis (SwYG) strain grew ca. 20% slower than its ancestor
with the same glycolytic genes with native glycolysis design. Exploring the
cause of this slower growth rate, the present study reflects on the genetic and
analytical challenges encountered by extensive strain construction programs and
provides design guidelines for integration of large constructs in the yeast genome.
This study also suggests a potential involvement of the yeast glycolytic enzyme
phosphoglycerate kinase (Pgk1) in
PI(3)P synthesis and autophagy, as found in mammalian cells.
Includes supplemental figures
and tables with descriptions of their content.
OrganizationsTU Delft, Faculty of Applied Sciences, Department of Biotechnology