Physiological and Metabolic Challenges of Flocculating Saccharomyces cerevisiae in D-Lactic Acid Fermentation under High Glucose and Inhibitory Conditions

This study investigates two genetically engineered Saccharomyces cerevisiae F118 strains, ΔCYB2::LpDLDH and ΔPDC1::LpDLDH, for D-LA (D-LA) production under high-glucose and inhibitor-stress conditions that mimic lignocellulosic hydrolysates. At 150 g/L glucose, ΔCYB2::LpDLDH produced 60 g/L D-LA, whereas ΔPDC1::LpDLDH yielded 80 g/L, corresponding to 40% and 53% of the theoretical yield, respectively. Calcium carbonate (CaCO3) supplementation enhanced glucose consumption and strengthened flocculation in ΔPDC1::LpDLDH. The addition of 5% inhibitory chemical compounds (ICCs) redirected carbon flux in ΔCYB2::LpDLDH toward D-LA formation and reduced ethanol byproduct accumulation. Transcriptomic analysis revealed upregulation of stress-response genes (HOG1, TPS1) and cell wall remodeling genes (CRH1, SCW10) under high-glucose stress. The strongly flocculent F118ΔCYB2::LpDLDH strain exhibited greater tolerance to weak acids and furfural than the weakly flocculent F118ΔPDC1::LpDLDH strain, likely due to physical shielding and cooperative detoxification within flocs. Metabolomic profiling indicated that under inhibitor stress, carbon flux was diverted from the TCA cycle toward lactate synthesis to maintain redox balance. These findings highlight the multifaceted benefits of flocculation in enhancing strain robustness and D-LA productivity under harsh fermentation environments, providing insights for developing resilient yeast platforms for lignocellulosic bioprocessing.