Modulation of the general stress response impacts protein expression, energy economy, and heterogeneity in virtually non-dividing yeast cultures

Microbial cells adapt their physiology when growth is restricted by nutrient limitation, balancing survival with the capacity to produce proteins. Here, we investigated this adaptation in Saccharomyces cerevisiae by comparing retentostat cultures, in which specific growth rates reached below 0.001 h⁻¹, of a reference strain (ScNTi001) and a ∆ msn2 ∆ msn4 mutant (ScNTm001). We show that general stress-response transcription factors Msn2 and Msn4 are crucial for maintaining energy efficiency and for survival under this extreme calorie restriction. Their absence increased population heterogeneity and reduced viability, underscoring a role beyond merely stress resistance. To understand (heterologous) protein expression dynamics, we monitored how a stress-inducible promoter (P HSP12 ) and a constitutive promoter (P TEF1 ) impacted protein levels. The P HSP12 remained active at moderate to low growth rates, but expression sharply dropped below 0.001 h⁻¹, whereas the P TEF1 sustained protein expression, with activity linked to specific glucose uptake rates rather than growth rate. This decoupling revealed a trade-off: although P TEF1 -driven ymNeongreen-specific production rates were 70% higher in the absence of ∆ msn2 ∆ msn4 , overall product yields were 13% lower. IMPORTANCE This study demonstrates how Msn2 and Msn4 govern cellular robustness under extreme nutrient-limited conditions at near-zero growth rates. It also guides promoter selection strategies for growth-independent production processes. More broadly, this study shows that retentostats can be used as a valuable tool to dissect stress-response pathways, survival mechanisms, and growth-independent expression systems in microbial cell factories.