Integrating processes: Impact of upstream conditions on cell wall properties and intracellular protein release from mildly disrupted yeast

Cultivation conditions are known to affect the structural organization of microbial cell walls. However, the impact of these structural variations on protein release following mild cell disruption remains largely unknown. Therefore, this study investigated the effects of cultivation conditions on the Saccharomyces cerevisiae cell wall. Its robustness and apparent protein permeability were assessed indirectly through, respectively, zymolyase susceptibility and intracellular protein release following hydrophobic deep eutectic solvent assisted membrane-permeabilization. More specifically, the influences of cultivation pH, glucose-limitation, growth phase and cultivation mode were evaluated. Shake-flask experiments demonstrated that cultivation conditions associated with different physiological states strongly affected cell wall structural organization, since a shift from glucose-limitation to glucose-abundance increased zymolyase susceptibility by 5-fold, and increased protein release by up to 3.8-fold. Therefore, the effects of growth phase and cultivation mode were further investigated in controlled bioreactors, resulting in more profound differences. Biomass harvested from exponentially growing batch cultures exhibited a 5.7-fold higher soluble protein release than low-rate fed-batch cultures, while zymolyase susceptibility increased by 19.4-fold. Moreover, strong correlations were found between physiological state, zymolyase susceptibility and protein permeation behaviour, suggesting that cultivation-dependent physiological states impact cell wall characteristics and intracellular protein release. Finally, apparent molecular size analysis demonstrated that the released material obtained from permeabilized biomass was enriched with components smaller than 100 kDa. Overall, this study demonstrates that cultivation-driven modulations in cell wall characteristics strongly affect mild disruption yields, thereby highlighting the importance of integrating upstream and downstream processes for efficient recovery of intracellular proteins.