Engineering the acid-tolerant yeast Issatchenkia orientalis for efficient lactic acid production via optimized glucose and xylose utilization

Abstract

The acid-tolerant yeast Issatchenkia orientalis is a promising platform for the sustainable production of organic acids. However, the inefficient conversion of lignocellulosic biomass-derived sugars, primarily due to carbon catabolite repression (CCR), reduces overall production efficiency and limits its industrial application. In this study, we established a targeted genetic framework for efficient glucose–xylose co-utilization by coordinating hexokinase (HXK) modulation and transport-level engineering. Sequential fed-batch fermentations revealed that a xylose-initiated feeding strategy achieved a 2.04-fold higher lactic acid yield than simultaneous fermentation. To bypass carbon catabolite repression, endogenous hexokinases were characterized, and single deletions (hxk1Δ, hxk2Δ, or hxk3Δ) were conducted to attenuate glucose dominance. While this approach improved lactic acid yields, it simultaneously imposed severe kinetic bottlenecks. To address these limitations, heterologous sugar transporters, plant-derived AtSWEET7 and yeast-derived LST1, were integrated. Characterization in the intact background revealed that the Major Facilitator Superfamily (MFS)-type LST1 from Lipomyces starkeyi outperformed AtSWEET7. Double-copy integration of LST1 yielded the engineered SD108XL-LST2 strain, which achieved a lactic acid titer of 53.4 g/L within 56 h from a mixed-sugar substrate containing approximately 45 g/L glucose and 44 g/L xylose. Notably, the final yield (0.63 g/g) and volumetric productivity (0.96 g/L·h) represented 57.5% and 47.7% increases over the parental SD108XL strain, respectively. This transport-driven strategy effectively overrides native metabolic hierarchies while preserving essential glycolytic signaling, offering a robust framework for high-efficiency lignocellulosic biorefineries for organic acid production.