Lactic acid is an important biobased chemical widely used in the production of biodegradable plastics, food, and pharmaceuticals. However, the application of flocculant
Saccharomyces cerevisiae remains limited in addressing stresses such as high-glucose and inhibitor-rich conditions derived from biomass, particularly in D-lactic acid
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Lactic acid is an important biobased chemical widely used in the production of biodegradable plastics, food, and pharmaceuticals. However, the application of flocculant
Saccharomyces cerevisiae remains limited in addressing stresses such as high-glucose and inhibitor-rich conditions derived from biomass, particularly in D-lactic acid (D-LA) production. This study investigates two genetically engineered
S. cerevisiae F118 strains, Δ
CYB2::
LpDLDH and Δ
PDC1::
LpDLDH, for D-LA production under high-glucose and inhibitor-stress conditions that mimic lignocellulosic hydrolysates in shake-flask fermentation. At 150 g/L glucose, Δ
CYB2::
LpDLDH produced 41 ± 0.73 g/L D-LA, whereas Δ
PDC1::
LpDLDH yielded 80 ± 1.78 g/L, corresponding to 27% and 53% of the theoretical yield, respectively. Calcium carbonate (CaCO
3) supplementation enhanced glucose consumption and strengthened flocculation in Δ
PDC1::
LpDLDH. The addition of 5% inhibitory chemical compounds (ICCs) consisting of furfural, HMF, and weak acids redirected carbon flux in Δ
CYB2::
LpDLDH toward D-LA formation and reduced ethanol byproduct accumulation. Transcriptomic analysis revealed the upregulation of stress-response genes (HOG1, TPS1) and cell-wall remodeling genes (
CRH1,
SCW10) in response to high-glucose stress. The strongly flocculent F118Δ
CYB2::
LpDLDH strain exhibited greater tolerance to weak acids and furfural than the weakly flocculent F118Δ
PDC1::
LpDLDH strain. 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.
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