Findings indicate that yeast robustness can emerge through distinct adaptive strategies shaped by the genetic background, involving transcriptional rewiring or morphogenetic adaptation to lignocellulosic stress.
Abstract
Microbial robustness (i.e., to keep the same performance in the face of several perturbations) is a desirable trait for industrial yeasts, particularly in second-generation bioethanol production, where inhibitory compounds in lignocellulosic hydrolysates impair microbial performance. In this study, we investigated the role of the stress-responsive kinase Rim15 in stress adaptation and robustness by subjecting Saccharomyces cerevisiae wild-type (WT) and rim15Δ strain to adaptive laboratory evolution (ALE) in synthetic spruce hydrolysate (SSH). This hydrolysate contains high concentrations of inhibitory compounds, such as acetic acid and 5-hydroxymethylfurfural. Crucially, ALE consisted of two steps, including a medium swap regime that enabled adaptation to 100% SSH. Although WT and rim15Δ strain followed distinct evolutionary trajectories during ALE, both strains improved growth performance and robustness on SSH. The ability of the rim15Δ strain to adapt to 100% SSH was associated with impaired daughter cell separation and multicellular clump formation. Genome sequencing identified a mutation in a morphogenetic gene (ACE2) in Evolved rim15Δ. This differed from mutations in key transcriptional regulators (e.g., SSN8, SSN2) identified in Evolved WT. Reverse engineering indicated that ACE2 loss-of-function contributes to the multicellular clumping phenotype of the Evolved rim15Δ strain, while deletion mutants of SSN2 and SSN8 exhibited improved growth in SSH compared to the parental strain. Together, these findings indicate that yeast robustness can emerge through distinct adaptive strategies shaped by the genetic background, involving transcriptional rewiring or morphogenetic adaptation to lignocellulosic stress.
Saccharomyces cerevisiae is an essential fermentation ingredient in the brewing industry, where its genomic information and fermentation traits profoundly influence final product characteristics. Here, a comprehensive phenotypic and genomic characterisation of the ale yeast strain (CGMCC 2.0002) was performed under simulated brewing conditions. Microscopic observations revealed that the strain exhibits an ellipsoidal morphology with smooth cell surfaces. During brewing, fermentation at 20 °C resulted in accelerated sugar depletion and significantly higher alcohol production compared with fermentation at 11 °C, although its flocculation properties were weaker at the higher temperature. Sensory evaluation indicated that the beer produced at 20 °C exhibited improved colour and taste. Meanwhile, the concentrations of ethyl acetate, isoamyl acetate, catechin, and salicylic acid in the beer all increased. Whole-genome resequencing using the S. cerevisiae S288C reference genome identified 42 711 single nucleotide polymorphisms, 4 725 insertions and deletions, and 562 structural variations in ale yeast. Notably, 21 mutated loci were situated within the essential genes of the glycolytic pathway. These findings establish a solid genomic and physiological baseline for optimising the application of this strain in craft beer production.
Yong-Peng Jia, Hui-Xing Li, Bin Xu et al.· Journal of Food and Nutritio...· 0 citations
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.
Rebecca My, L. Corte, G. Cardinali et al.· Critical Reviews in Biotechn...· 0 citations
Xylitol is a sugar alcohol of interest in the food, pharmaceutical, and healthcare industries due to its applications as a food sweetener and sugar substitute. A wild-type Saccharomyces cerevisiae yeast strain designated 202-3 was isolated from a Colombian distillery located near sugarcane fields. This diploid 202-3 strain showed non-common, modest but noticeable xylose consumption and xylitol production in lignocellulosic hydrolysates. To enhance its natural xylose consumption ability, the strain was genetically engineered and submitted to adaptive laboratory evolution (ALE). Firstly, it was considered the deletion of the GAL80 gene to enable continuous expression of GAL genes, enhancing the uptake and assimilation of xylose. While the deletion of one copy of GAL80 (strain 202-3/∆) showed improved xylose consumption and xylitol production, better results were obtained when both copies of GAL80 were silenced (strain 202-3/∆∆). Subsequently, ALE experiments were conducted for these three strains in rich medium containing 20 g/L xylose. While the parental 202-3 strain consumed 2.46 g/L xylose and produced 0.42 g/L xylitol, the evolved 202-3/∆∆/ALE strain was able to consume 5.61 g/L xylose and produced 4.87 g/L xylitol, with a xylitol yield of 0.87 g xylitol/g xylose, and also the highest xylitol volumetric productivity (0.034 g xylitol/L/h) among the strains. Thus, our engineered and evolutionary experiments allowed a significant improvement in terms of xylose consumption, xylitol production and xylitol yield.
Margareth Andrea Patiño Lagos, Diana Carolina Tusso Pinzón, Jorge Alejandro Cristancho Caviativa et al.· Fermentation· 0 citations
Furfural and 5-hydroxymethylfurfural (HMF) are two major lignocellulosic growth inhibitors that hinder microbial growth and fermentation of lignocellulosic hydrolysate for lactic acid production. In this study, we employed adaptive laboratory evolution (ALE) to enhance the tolerance of Lactiplantibacillus plantarum JGR2, a strain previously isolated in our lab, to furfural and HMF. The adapted strains demonstrated significantly improved growth in the presence of these inhibitors compared to the parental strains. Whole-genome resequencing revealed multiple mutations including high-impact non-conservative mutations in genes encoding DNA recombination and repair protein (RecF, lp_0005), flavin prenyltransferase (UbiX lp_0271), and oligo-1,6-glucosidase (lp_0189). Transcriptomic analysis indicated that adaptation elicited more pronounced differential gene expression compared to acute inhibitor exposure. Upon furfural exposure, the furfural-adapted isolate showed fewer differentially expressed genes than the parental strain, indicating a possible shift in the transcriptomic profile as a possible mechanism of furfural adaptation. Mechanistic investigation revealed that the adapted isolates reduce furfural into the less toxic furfuryl alcohol, suggesting a key detoxification mechanism. Notably, lp_3051 (dhaT, 1,3-propanediol dehydrogenase) encoding furfural reductase activity was upregulated in both furfural- and HMF-adapted isolates. Membrane fatty acid analysis revealed increased unsaturated fatty acids and cyclopropane fatty acids in adapted strains. Finally, the adapted strains exhibited improved growth in rice straw hydrolysate and produced significantly higher relative lactic acid yields compared to the parental strain, thus demonstrating improved bioproduction under inhibitor-rich conditions. This study not only provides a comprehensive understanding of L. plantarum’s response to lignocellulosic inhibitors, but also yields evolved bacterial candidates for further scientific and industrial exploration.
Sharoni Sharma, Sarvesh V. Surve, J. Fernandes et al.· Bioresources and Bioprocessi...· 0 citations
Adaptive laboratory evolution (ALE) was successfully applied to improve the tolerance of Saccharomyces cerevisiae toward butyric acid, enabling its use in co-culture with Clostridium tyrobutyricum for the simultaneous production of ethanol and butyric acid as ester precursors. S. cerevisiae was adapted through serial transfer at progressively increasing butyric acid concentrations up to 20 g L⁻1. The evolved yeast population exhibited significantly enhanced butyric acid tolerance and maintained ethanol production under acid-stress conditions. Interestingly, the evolved population also displayed increased maximum glucose consumption rate and ethanol productivity under non-stress conditions. Whole-genome variant analysis was performed by comparing the wild-type strain, an intermediate evolved population and the final evolved population obtained during ALE. The results suggest that adaptation may be associated with changes in regulatory processes, cellular homeostasis, and membrane and cell wall remodeling. The co-culture with the acid producer C. tyrobutyricum demonstrated efficient and balanced substrate utilization, indicating a stable division of labor between the two organisms. Fed-batch cultivation yielded 20.31 ± 2.43 g L⁻1 butyric acid and 28.18 ± 3.37 g L⁻1 ethanol. With the evolved yeast, ethanol concentrations increased fourfold compared to those achieved with the wild-type strain in previous studies, underscoring the potential of ALE to overcome limitations in co-culture systems.
K. Oehlenschläger, S. Bauschatz, E. Schepp et al.· Microbial Cell Factories· 0 citations
Methanol is a reduced, soluble one-carbon (C1) feedstock for sustainable bioproduction, but converting this potential into robust microbial growth remains difficult. Several synthetic C1 assimilation routes depend on autocatalytic cycles, whose operation requires coordinated control of redox balance, toxic intermediates, substrate regeneration, and host regulation. Here, we implemented the serine-threonine cycle (STC) in the soil bacterium Pseudomonas putida and used growth-coupled selection with adaptive laboratory evolution (ALE) to transition from mixotrophic C1 incorporation to strict methylotrophy. The evolved strain grew with methanol as the sole carbon and energy source under atmospheric CO2 with a doubling time of ca. 40 h. Whole-genome sequencing, reverse genetics, biosensors, isotope labelling, and comparative RNA sequencing showed that evolution repeatedly targeted native pyrroloquinoline quinone (PQQ)-dependent methanol oxidation, membrane-bound transhydrogenase activity, glycine regeneration, STC enzyme balance, and global regulatory nodes. Additional ALE under glycine-methanol co-feeding increased growth rates and exposed further targets for improving cycle flux. These results establish P. putida as a chassis for strict synthetic methylotrophy and define actionable engineering routes toward C1 biomanufacturing. GRAPHICAL ABSTRACT
Òscar Puiggené, Martina Fricano, Riccardo Rossi et al.· bioRxiv· 0 citations