Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae

Abstract

Chemostat cultivation mode imposes selective pressure on the cells, which may result in slow adaptation in the physiological state over time. We applied a two‐compartment scale‐down chemostat system imposing feast–famine conditions to characterize the long‐term (100 s of hours) response of Saccharomyces cerevisiae to fluctuating glucose availability. A wild‐type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale‐down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared with those observed in a well‐mixed chemostat. Our results show that S. cerevisiae subjected to long‐term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast–famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hr. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms.

Single-Cell Technologies to Understand the Mechanisms of Cellular Adaptation in Chemostats

“…Further development of the technologies are therefore needed. Adaptation in chemostats affects both the genome, transcriptome, metabolome, and proteome (Dunham et al, 2002;Kazemi Seresht et al, 2013;Wright et al, 2020). We envision the application of single-cell omics for the holistic study of the adaptive mechanisms, as the omics technologies have the potential to measure large amounts of parameters at all regulatory levels.…”

“…Moreover, decreased (over)capacity of the main carbon metabolism including the glycolysis and TCA cycle is observed and has been suggested as a way to get an energetical advantage (Mashego et al, 2005). Cellular stress-responses are in many cases also differentially expressed between early and late cultivation stages including proteins involved in heat shock, oxidative stress, and damage resistance (Jansen et al, 2005;Franchini and Egli, 2006;Wright et al, 2020). Morphological changes toward filamentous and pseudo-hyphal growth are known effects of chemostat growth (Brown and Hough, 1965;Adams et al, 1985;Rebnegger et al, 2014;Rai et al, 2019) and also a known adaptive response to nutrient poor environments (Gimeno et al, 1992).…”

“…Morphological changes toward filamentous and pseudo-hyphal growth are known effects of chemostat growth (Brown and Hough, 1965;Adams et al, 1985;Rebnegger et al, 2014;Rai et al, 2019) and also a known adaptive response to nutrient poor environments (Gimeno et al, 1992). The productivity of industrially relevant strains often decreases over time during chemostat growth (Douma et al, 2011;Paulová et al, 2012;Kazemi Seresht et al, 2013;Wright et al, 2016Wright et al, , 2020. A reduced productivity can be construed as a clear growth advantage over cells that are not able to reduce the burden of heterologous production.…”

“…Whether and when a given mutation will dominate a culture depends on the relative fitness of the mutant compared to other clones in the population (Gresham and Hong, 2015). For industrial strains, studies report reproducible adaptive changes in transcriptome, proteome, and heterologous product already after 22 generations of chemostat growth (Douma et al, 2011;Kazemi Seresht et al, 2013;Wright et al, 2020). The observed changes cannot always be coupled to genetic instability (Douma et al, 2011) and may therefore be related to other adaptive mechanisms, e.g., epigenetics.…”

The future of self-selecting and stable fermentations

“…Observed at bulk-population level, changes in protein-composition during long-term cultivation e.g. affected insulin precursor production in S. cerevisiae expressed from a multi-copy plasmid [34,64]. In the case of ethanol-producing S. cerevisiae, short-term adaptation to the medium during propagation improves the quality of Five hypothetical heterogeneity scenarios are presented in relation to, a bulkpopulation performance, b bulk population-specific growth rate and c) population composition with regards to producing cells (colored), low-producing (gray-colored) and non-producing cells (blackcolored).…”