Performing in spite of starvation: How Saccharomyces cerevisiae maintains robust growth when facing famine zones in industrial bioreactors

Abstract

In fed‐batch operated industrial bioreactors, glucose‐limited feeding is commonly applied for optimal control of cell growth and product formation. Still, microbial cells such as yeasts and bacteria are frequently exposed to glucose starvation conditions in poorly mixed zones or far away from the feedstock inlet point. Despite its commonness, studies mimicking related stimuli are still underrepresented in scale‐up/scale‐down considerations. This may surprise as the transition from glucose limitation to starvation has the potential to provoke regulatory responses with negative consequences for production performance. In order to shed more light, we performed gene‐expression analysis of Saccharomyces cerevisiae grown in intermittently fed chemostat cultures to study the effect of limitation‐starvation transitions. The resulting glucose concentration gradient was representative for the commercial scale and compelled cells to tolerate about 76 s with sub‐optimal substrate supply. Special attention was paid to the adaptation status of the population by discriminating between first time and repeated entry into the starvation regime. Unprepared cells reacted with a transiently reduced growth rate governed by the general stress response. Yeasts adapted to the dynamic environment by increasing internal growth capacities at the cost of rising maintenance demands by 2.7%. Evidence was found that multiple protein kinase A (PKA) and Snf1‐mediated regulatory circuits were initiated and ramped down still keeping the cells in an adapted trade‐off between growth optimization and down‐regulation of stress response. From this finding, primary engineering guidelines are deduced to optimize both the production host's genetic background and the design of scale‐down experiments.

Mimicked Mixing-Induced Heterogeneities of Industrial Bioreactors Stimulate Long-Lasting Adaption Programs in Ethanol-Producing Yeasts

“…Anaerobic cultivation experiments were carried out in a stainless-steel benchtop bioreactor (Bioengineering, Wald, Switzerland) with a liquid working volume of 1.7 l under a 0.3 bar overpressure. The reactor system and its rapid sampling device were operated as previously described [15,23] with the following modifications: (i) silicone tubing was replaced by oxygen-impermeable tubing (Norprene, Cole Parmer, Vernon Hills, IL, USA), (ii) anaerobiosis was maintained with a sterile nitrogen supply of 0.425 vvm and (iii) no antifoam agent was supplied as it was already present in the medium. Furthermore, the headspace of the feed casket was kept flushed with sterile nitrogen throughout the experiment.…”

“…Sample processing and analysis are thoroughly reported in [15] and [23]. In brief, biomass, expressed as dry matter of biomass (DMB), was determined gravimetrically.…”

“…Transcriptional responses of ScER were compared to the likewise stimulated haploid MATa strain CEN.PK 113-7D [23] that was growing under aerobic conditions (Figure 8). Except for different |log 2 -fold change| thresholds, the same analytical pipeline was used in both studies.…”

“…Taken together, we interpret the overall sparsity of common gene sets across conditions per strain as further evidence of a highly conserved transcriptional regulation circuitry, which operates at the glucose limitation-starvation junction. [23] conditions in LSL-cycling chemostats. Black dots in the matrix indicate the considered DEG sets, the bar above represents the respective intersection size and the color gradient illustrates significance.…”

“…Derived knowledge on the gene-regulation level ultimately guided rational strain engineering approaches to increase microbial robustness [21,22]. In a recent study, we investigated the transcriptional profile of respiring S. cerevisiae against short-term transitions between glucose limitation and starvation in an analogous approach [23]. First-time exposure to acute glucose depletion elicited the ESR prematurely in a non-adapted culture, while it was globally repressed in a 'stand-by mode' enabling dynamic response once the population was adapted to the signals.…”