Integrated Physiological and Transcriptomic Analyses of Saccharomyces cerevisiae Under Syringaldehyde Stress

Harnessing natural diversity of Saccharomyces cerevisiae for enhanced resistance to weak acids

Abstract The production of second-generation bioethanol from lignocellulosic biomass is a promising solution for sustainable energy, yet it faces significant challenges also due to the inhibitory effects of weak acids released during biomass pretreatment, particularly acetic, formic and levulinic acids. This review describes the ability of Saccharomyces cerevisiae, with a focus on natural isolates, in overcoming these challenging compounds. Indeed, natural isolates exhibit greater genetic and phenotypic diversity than laboratory and industrial strains, offering unique traits such as enhanced stress tolerance, metabolic efficiency, and adaptive responses to weak acids. This investigation explores the transcriptional and genomic mechanisms underlying yeast adaptive responses, emphasizing key regulatory networks and resistance pathways, including drug H+ antiporters, Reactive Oxygen Species (ROS) mitigation strategies, and membrane composition adjustments. Strategies for strains improvement, involving adaptive laboratory evolution (ALE), genome shuffling, and hybridization, are also discussed as complementary approaches to develop robust yeast capable of thriving under stressful industrial fermentation conditions. The integration of these techniques, along with genomic and transcriptomic insights, provides a comprehensive framework for engineering high-performance yeast strains. Ultimately, this review underscores the potential of leveraging natural diversity and innovative biotechnological strategies to advance the scalability and efficiency of lignocellulosic bioethanol production through S. cerevisiae fermentation. Graphical abstractMulti-panel diagram illustrating Saccharomyces cerevisiae isolates, genomic exploration, and metabolic engineering techniques.The figure features three connected panels detailing research on Saccharomyces cerevisiae. The first panel illustrates yeast cell icons with phenotypic traits: "High fermentative," "Weak acid resistant," "High secretory pathway," "Thermotolerant," and "Osmotic tolerant," with a glucose structure below. The second showcases a circular diagram of a "Mosaic genome," "Copy Number Variations," and single nucleotide polymorphisms (SNPs), with a heatmap for transcriptomics. The third illustrates metabolic engineering techniques, including CRISPR/Cas9, genome editing, Delta-integration, and transformation within yeast cells. STATEMENT OF SIGNIFICANCE Weak acids represent major inhibitory compounds in lignocellulosic fermentations, affecting the industrial viability of second-generation bioethanol. While most research has focused on laboratory or engineered strains, natural isolates of Saccharomyces cerevisiae can be still considered a resource of unexplored tolerance traits. This review highlights how omics-based insights into natural strains’ adaptive responses provide novel opportunities for metabolic engineering, particularly in weak acid resistance. By integrating genetic variability, ploidy diversity, and systems biology perspectives, a yeast strain capable of overcoming industrial-relevant stresses such as weak acids can be designed, advancing sustainable bioethanol production and expanding the scope of microbial biotechnology.

Curcumin Enhances Alcoholic Fermentation in Yeast by Promoting Osmotic Stress Adaptation

Spices contain diverse plant secondary metabolites with antimicrobial and antioxidant activities that can influence plant–microbe interactions. Among these metabolites, curcumin and piperine are major bioactive constituents of turmeric and black pepper, respectively. In this study, we demonstrate that curcumin and piperine enhance alcoholic fermentation in the budding yeast Saccharomyces cerevisiae. Curcumin significantly promoted alcoholic fermentation under high-glucose conditions (20–40% glucose), which impose substantial osmotic stress on yeast cells. Under these conditions, curcumin enhanced yeast growth, improved osmotic stress tolerance, and increased resistance to cell wall digestion, suggesting enhanced stress adaptation during fermentation. Furthermore, deletion of HOG1, which encodes the central MAP kinase of the High-Osmolarity Glycerol (HOG) pathway responsible for the osmotic stress response, abolished the fermentation-enhancing effect of curcumin, indicating that Hog1 function is required for this effect. Consistent with this result, transcriptomic and RT- qPCR analyses revealed increased expression of stress adaptation-related genes, including known HOG pathway-responsive genes. In contrast, the expression of genes involved in amino acid catabolism was decreased at 12 h after the initiation of fermentation, while metabolomic analysis revealed increased intracellular levels of amino acids, including glutamate and asparagine, at 24 h. These coordinated transcriptional and metabolic changes are consistent with reduced amino acid catabolism and a shift toward a metabolic state favorable for sustained fermentation. Together, these findings suggest that curcumin enhances alcoholic fermentation under osmotic stress by promoting stress adaptation that supports yeast growth and sustained fermentation. Importance Plant secondary metabolites are widely recognized for their roles in plant defense, whereas their beneficial effects on microbial physiology and metabolism remain poorly understood. This study demonstrates that the spice-derived compounds curcumin and piperine enhance alcoholic fermentation in the budding yeast Saccharomyces cerevisiae. Curcumin, in particular, improved yeast growth and stress tolerance under high-glucose conditions that impose substantial osmotic stress. Its fermentation-enhancing effect required Hog1 function and was accompanied by increased expression of HOG pathway- responsive genes and changes in amino acid metabolism. These findings demonstrate that plant-derived compounds can not only exert antimicrobial effects but also beneficially modulate yeast stress adaptation and fermentation performance. The use of natural bioactive compounds to improve fermentation robustness under high-sugar conditions may contribute to increased productivity and reduced costs in fermented food and bioethanol production.

Transcriptomic Analysis Reveals How PHO4 Gene Modulates Growth and Ethanol Fermentation in Saccharomyces cerevisiae

PHO4 is identified as a promising candidate target for improving high concentration ethanol fermentation efficiency and provides a framework to understand the phosphate-dependent regulatory effects of PHO4 allelic variation and offer a transferable strategy for strain improvement.

Integrating Morphology and Transcriptional Regulation to Alleviate Intracellular Product Stress for Enhanced Indigo Biosynthesis in Escherichia coli.

Indigo is an important biobased colorant used in textile dyeing and as a precursor for blue food colorants; however, its microbial production is often limited by cytotoxicity and intracellular accumulation of insoluble products. Here, we established a systems-level host engineering framework in Escherichia coli to improve indigo biosynthesis. CRISPRi screening of cell envelope-associated genes identified targets including sulA, ftsZ, fabF, and pgi. Mechanistic analyses showed that modulation of cell division and central carbon metabolism alleviates envelope stress and enhances reducing power for indigo formation, while electron microscopy confirmed reduced cellular perturbation caused by intracellular indigo accumulation. A second CRISPRi library targeting transcription factors revealed coordinated regulation of BolA, ArcA, and RpoN to balance metabolism and stress responses. Integration of targets enabled 9.38 g/L indigo production in a 5 L fed-batch bioreactor, a 2.55-fold improvement over the parental strain. This work provides a promising engineering strategy for microbial indigo production.

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.

Regulating microbial cell factories to resist ROS stress in chemical production