When faced with environmental changes, microbes often enter a temporary growth arrest during which they reprogram the expression of specific genes to adapt to the new conditions. A prime example of such a lag phase occurs when microbes need to switch from glucose to other, less-preferred carbon sources. Despite its industrial relevance, the genetic network that determines the duration of the lag phase has not been studied in much detail. Here, we performed a genome-wide Bar-Seq screen to identify genetic determinants of the Saccharomyces cerevisiae glucose-to-galactose lag phase. The results show that genes involved in respiration, and specifically those encoding complexes III and IV of the electron transport chain, are needed for efficient growth resumption after the lag phase. Anaerobic growth experiments confirmed the importance of respiratory energy conversion in determining the lag phase duration. Moreover, overexpression of the central regulator of respiration, HAP4, leads to significantly shorter lag phases. Together, these results suggest that the glucose-induced repression of respiration, known as the Crabtree effect, is a major determinant of microbial fitness in fluctuating carbon environments.IMPORTANCE The lag phase is arguably one of the prime characteristics of microbial growth. Longer lag phases result in lower competitive fitness in variable environments, and the duration of the lag phase is also important in many industrial processes where long lag phases lead to sluggish, less efficient fermentations. Despite the immense importance of the lag phase, surprisingly little is known about the exact molecular processes that determine its duration. Our study uses the molecular toolbox of S. cerevisiae combined with detailed growth experiments to reveal how the transition from fermentative to respirative metabolism is a key bottleneck for cells to overcome the lag phase. Together, our findings not only yield insight into the key molecular processes and genes that influence lag duration but also open routes to increase the efficiency of industrial fermentations and offer an experimental framework to study other types of lag behavior.

Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of "pathway swapping," using yeast glycolysis as the experimental model. Construction of a "single-locus glycolysis" Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast's entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.