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The response mechanism analysis of HMX1 knockout strain to levulinic acid in Saccharomyces cerevisiae.

ABSTRACT: Levulinic acid, a hydrolysis product of lignocellulose, can be metabolized into important compounds in the field of medicine and pesticides by engineered strains of Saccharomyces cerevisiae. Levulinic acid, as an intermediate product widely found in the conversion process of lignocellulosic biomass, has multiple applications. However, its toxicity to Saccharomyces cerevisiae reduces its conversion efficiency, so screening Saccharomyces cerevisiae genes that can tolerate levulinic acid becomes the key. By creating a whole-genome knockout library and bioinformatics analysis, this study used the phenotypic characteristics of cells as the basis for screening and found the HMX1 gene that is highly sensitive to levulinic acid in the oxidative stress pathway. After knocking out HMX1 and treating with levulinic acid, the omics data of the strain revealed that multiple affected pathways, especially the expression of 14 genes related to the cell wall and membrane system, were significantly downregulated. The levels of acetyl-CoA and riboflavin decreased by 1.02-fold and 1.44-fold, respectively, while the content of pantothenic acid increased. These findings indicate that the cell wall-membrane system, as well as the metabolism of acetyl-CoA and riboflavin, are important in improving the resistance of Saccharomyces cerevisiae to levulinic acid. They provide theoretical support for enhancing the tolerance of microorganisms to levulinic acid, which is significant for optimizing the conversion process of lignocellulosic biomass to levulinic acid.

SUBMITTER: Tang J

PROVIDER: S-EPMC11233763 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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The response mechanism analysis of <i>HMX1</i> knockout strain to levulinic acid in Saccharomyces cerevisiae.

Tang Jiaye J Chen Yulei Y Li Qian Q Xin Wenli W Xiao Ximeng X Chen Xuemei X Yang Lixi L Mou Borui B Li Jialian J Lu Fujia F Fu Chun C Long Wencong W Liao Hong H Han Xuebing X Feng Peng P Li Wei W Zhou Kedi K Yang Liuyun L Yang Yaojun Y Ma Menggen M Wang Hanyu H

Frontiers in microbiology 20240626

Levulinic acid, a hydrolysis product of lignocellulose, can be metabolized into important compounds in the field of medicine and pesticides by engineered strains of Saccharomyces cerevisiae. Levulinic acid, as an intermediate product widely found in the conversion process of lignocellulosic biomass, has multiple applications. However, its toxicity to Saccharomyces cerevisiae reduces its conversion efficiency, so screening Saccharomyces cerevisiae genes that can tolerate levulinic acid becomes th ...[more]

PMID: 38989024

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Project description:Sugarcane molasses is an ideal economical raw material for ethanol production because of its wide availability, low cost and nutrient content. However, benzoic acid compounds with toxic effects on yeast cells are commonly found in sugarcane molasses. At present, the molecular mechanism of the toxic effects of benzoic acid on Saccharomyces cerevisiae has not been elucidated. Here, the toxic effect of exogenous benzoic acid on S. cerevisiae GJ2008 cells was studied, and the genes differentially expressed in S. cerevisiae GJ2008 after 1.2 g/L benzoic acid stress were identified via Illumina RNA-Seq technology. The results indicated that benzoic acid significantly inhibited yeast cell growth, prolonged their rapid growth period, and ultimately reduced their biomass. During ethanol fermentation using 250 g/L sucrose under 1.2 g/L benzoic acid stress, several adverse effects were observed, such as high residual sugar content, low ethanol concentration and low fermentation efficiency. In addition, the cell morphology was damaged, the cell membrane permeability increased, intracellular nucleic acid and protein leakage increased, and the malondialdehyde content significantly increased. Moreover, the cells protected themselves by significantly increasing the intracellular glycerol content. Fourier transform infrared spectroscopy proved that benzoic acid could reduce the degree of unsaturation and increase cell membrane permeability by changing the yeast cell wall and cell membrane composition, leading to cell damage and even death. Transcriptomic analysis revealed that under benzoic acid stress, the expression of genes associated with sucrose and starch metabolism, thiamine metabolism, the glycolysis pathway, fructose and mannose metabolism, galactose metabolism and ABC transporters was significantly downregulated. The expression of genes related to ribosomes, lipid metabolism, ribosome biosynthesis, nucleic acid metabolism, arginine and proline metabolism, RNA polymerase, metabolism related to cofactor synthesis, and biosynthesis of valine, leucine, and isoleucine was significantly upregulated. These results indicated that benzoic acid inhibited glycolysis and reduced sugar absorption and utilization and ATP energy supply in yeast cells. In response to stress, genes related to the ribosome bioanabolic pathway were upregulated to promote protein synthesis. On the other hand, the expression of ELO1, SUR4, FEN1 and ERG1 was upregulated, which led to extension of long-chain fatty acids and accumulation of ergosterol to maintain cell membrane structure. In conclusion, this paper provides important insights into the mechanism underlying the toxicity of benzoic acid to yeast cells and for realizing high-concentration ethanol production by sugarcane molasses fermentation.

Project description:BackgroundContinuous fermentation offers advantages in improving production efficiency and reducing costs, making it highly competitive for industrial ethanol production. A key requirement for Saccharomyces cerevisiae strains used in this process is their tolerance to high ethanol concentrations, which enables them to adapt to continuous fermentation conditions. To explore how yeast cells respond to varying levels of ethanol stress during fermentation, a two-month continuous fermentation was conducted. Cells were collected at different ethanol concentrations (from 60 g/L to 100 g/L) for comparative transcriptomic analysis.ResultsDuring continuous fermentation, as ethanol concentration increased, the expression of genes associated with cytoplasmic ribosomes, translation, and fatty acid biosynthesis progressively declined, while the expression of genes related to heat shock proteins (HSPs) and ubiquitin-mediated protein degradation gradually increased. Besides, cells exhibited distinct responses to varying ethanol concentrations. At lower ethanol concentrations (nearly 70 g/L), genes involved in mitochondrial ribosomes, oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, antioxidant enzymes, ergosterol synthesis, and glycerol biosynthesis were specifically upregulated compared to those at 60 g/L. This suggests that cells enhanced respiratory energy production, ROS scavenging capacity, and the synthesis of ergosterol and glycerol to counteract stress. At relatively higher ethanol concentrations (nearly 80 g/L), genes involved in respiration and ergosterol synthesis were inhibited, while those associated with glycolysis and glycerol biosynthesis were notably upregulated. This suggests a metabolic shift from respiration towards enhanced glycerol synthesis. Interestingly, the longevity-regulating pathway seemed to play a pivotal role in mediating the cellular adaptations to different ethanol concentrations. Upon reaching an ethanol concentration of 100 g/L, the aforementioned metabolic activities were largely inhibited. Cells primarily focused on enhancing the clearance of denatured proteins to preserve cellular viability.ConclusionsThis study elucidated the mechanisms by which an ethanol-tolerant S. cerevisiae strain adapts to increasing ethanol concentrations during continuous fermentation. The findings suggest that the longevity-regulating pathway may play a critical role in adapting to varying ethanol stress by regulating mitochondrial respiration, glycerol synthesis, ergosterol synthesis, antioxidant enzyme, and HSPs. This work provides a novel and valuable understanding of the mechanisms that govern ethanol tolerance during continuous fermentation.

Dataset Information

The response mechanism analysis of HMX1 knockout strain to levulinic acid in Saccharomyces cerevisiae.

Publications

The response mechanism analysis of <i>HMX1</i> knockout strain to levulinic acid in Saccharomyces cerevisiae.

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