What is wrong with SwYG? Reaching the limits of S. cerevisiae molecular and analytical toolbox

The 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, relocalized 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 percent slower than its ancestor 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.