Engineering cellular redox homeostasis to optimize ethanol production in xylose-fermenting Saccharomyces cerevisiae strains

The transition from fossil fuels dependency to embracing renewable alternatives is pivotal for mitigating greenhouse gas emissions, with biorefineries playing a central role at the forefront of this transition. As a sustainable alternative, lignocellulosic feedstocks hold great promise for biofuels and biochemicals production. However, the effective utilization of complex sugars, such as xylose, remains a significant hurdle. To address this challenge, yeasts can be engineered as microbial platforms to convert the complex sugars derived from biomass. The efficient use of xylose by XR-XDH strains still poses a significant challenge due to redox imbalance limitations, leading to the accumulation of undesirable by-products. In this study, we focused on engineering the industrial S. cerevisiae strain PE-2, known for its robustness, and compared different strategies to balance cellular redox homeostasis, guided by a genome-scale metabolic model. Flux balance analysis guided the selection of four approaches: i. decoupling NADPH regeneration from CO₂ production; ii. altering XDH cofactor affinity; iii. shifting XR cofactor preference; iv. incorporating alternate phosphoketolase and acetic acid conversion pathways. A comparative time-course targeted metabolic profile was conducted to assess the redox status of xylose-fermenting cells under anaerobic conditions. The main limitations of xylose-fermenting strains were tested and the replacement of xylose reductase with a NADH-preferred XR in the LVY142 strain proved to be the most effective strategy, resulting in an increase in ethanol yield and productivity, coupled with a reduction in by-products. Comparative analysis of various genetic approaches provided valuable insights into the complexities of redox engineering, highlighting the need for tailored strategies in yeast metabolic engineering for efficient biofuels and biochemicals production from lignocellulosic feedstocks.