Adaptive responses of Kluyveromyces marxianus CCT 7735 to 2-phenylethanol stress: Alterations in membrane fatty-acid composition, ergosterol content, exopolysaccharide production and reduction in reactive oxygen species

Balbino, Thércia Rocha; Silveira, Fernando A.; Ventorim, Rafaela Zandonade; Nascimento, Antônio Galvão do; Oliveira, Leandro Licursi de; Silveira, Wendel Batista da

doi:10.1016/j.fgb.2021.103561

Cited by 13 publications

(8 citation statements)

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“…2-PE was found to rapidly induce ROS accumulation of K. marxianus. Here, we found that 2-PE caused ROS accumulation in B. cinerea , triggering the activation of genes for antioxidant enzymes CAT, SOD, and POD and the upregulation of the sulfur and glutathione metabolism pathways (Figure C and Table ).…”

Section: Discussionmentioning

confidence: 72%

ROS Stress and Cell Membrane Disruption are the Main Antifungal Mechanisms of 2-Phenylethanol against Botrytis cinerea

Zou

Wei

Jiang

et al. 2022

J. Agric. Food Chem.

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2-Phenylethanol (2-PE), a common compound found in plants and microorganisms, exhibits broad-spectrum antifungal activity. Using Botrytis cinerea, we demonstrated that 2-PE suppressed mycelium growth in vitro and in strawberry fruit and reduced natural disease without adverse effects to fruit quality. 2-PE caused structural damage to mycelia, as shown by scanning and transmission electron microscopy. From RNA sequencing analysis we found significantly upregulated genes for enzymatic and nonenzymatic reactive oxygen species (ROS) scavenging systems including sulfur metabolism and glutathione metabolism, indicating that ROS stress was induced by 2-PE. This was consistent with results from assays demonstrating an increase ROS and hydrogen peroxide levels, antioxidant enzyme activities, and malondialdehyde content in treated cells. The upregulation of ATP-binding cassette transporter genes, the downregulation of major facilitator superfamily transporters genes, and the downregulation of ergosterol biosynthesis genes indicated a severe disruption of cell membrane structure and function. This was consistent with results from assays demonstrating compromised membrane integrity and lipid peroxidation. To summarize, 2-PE exposure suppressed B. cinerea growth through ROS stress and cell membrane disruption.

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Section: Discussionmentioning

confidence: 72%

ROS Stress and Cell Membrane Disruption are the Main Antifungal Mechanisms of 2-Phenylethanol against Botrytis cinerea

Zou

Wei

Jiang

et al. 2022

J. Agric. Food Chem.

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“…Alcohol 2-phenylethanol, produced by K. marxianus in moderate amounts, is the most well-studied quorum-sensing molecule in dimorphic yeasts (e.g., K. marxianus and S. cerevisiae ) [ 45 , 46 ]. Balbino et al showed that exposure of K. marxianus to 2-phenylethanol provoked changes in cell morphology and fermentative metabolism, and impaired yeast viability [ 47 ]. Isoamylalcohol is another signalling compound, which has been reported to induce pseudohyphal growth and filamentation in S. cerevisiae [ 48 ].…”

Section: Discussionmentioning

confidence: 99%

Spoilage Potential of Contaminating Yeast Species Kluyveromyces marxianus, Pichia kudriavzevii and Torulaspora delbrueckii during Cold Storage of Skyr

Srimahaeak

Petersen

Lillevang

et al. 2022

Foods

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This study investigated the spoilage potential of yeast strains Kluyveromyces marxianus (Km1, Km2 and Km3), Pichia kudriavzevii Pk1 and Torulaspora delbrueckii Td1 grown in skyr in cold storage. Yeast strains were isolated from skyr and identified by sequencing of the 26S rRNA gene. K. marxianus yeasts were grown in skyr to high numbers, generating large amounts of volatile organic compounds (VOC) associated with off-flavours, among them were alcohols (3-methyl-1-butanol, 2-methyl-1-propanol and 1-hexanol), esters (ethyl acetate and 3-methylbutyl acetate) and aldehydes (hexanal, methylbutanal and methylpropanal). Growth of P. kudriavzevii Pk1 led to moderate increases in several alcohols and esters (mostly, 3-methyl-1-butanol and ethyl acetate), whereas only minor shifts in VOCs were associated with T. delbrueckii Td2. The levels of the key aroma compounds, diacetyl and acetoin, were significantly decreased by all K. marxianus strains and P. kudriavzevii Pk1. In contrast to the other yeast species, K. marxianus was able to utilize lactose, producing ethanol and carbon dioxide. Based on the overall results, K. marxianus was characterised by the highest spoilage potential. The study revealed the differences between the yeast species in fermentative and spoilage activities, and clarified the role of yeast metabolites for off-flavour formation and quality defects in skyr during cold storage.

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“…The yeast cells were washed three times with PBS buffer and resuspended and diluted to an OD 600 of 0.2. Then the cell suspension was stained with propidium iodide (PI) to evaluate membrane integrity [ 25 ] and the oxidant-sensitive probe 2′,7′-dichlorofluorescein diacetate [ 6 ] for 30 min at 4 °C in the dark. Next, the stained samples were ultrasonicated for 1 min.…”

Section: Methodsmentioning

confidence: 99%

“…When the 2-PE concentration reached 2.5 g/L, the growth rate of S. cerevisiae decreased by 75% [ 1 ]. Additionally, an irregular change in cell morphology was observed in yeast subjected to 2-PE stress [ 6 ]. Transcriptome analyses suggested that 2-PE induced differences in the expression of genes associated with functions including mitochondrial activity, plasma membrane permeability, amino acid metabolism, and meiosis [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Evolutionary and reverse engineering in Saccharomyces cerevisiae reveals a Pdr1p mutation-dependent mechanism for 2-phenylethanol tolerance

Xia

Kang

et al. 2022

Microb Cell Fact

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Background 2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, inhibits growth of the producer strains. However, the limited knowledge regarding 2-PE tolerance mechanisms renders our current knowledge base insufficient to inform rational design. Results To improve the growth phenotype of Saccharomyces cerevisiae under a high 2-PE concentration, adaptive laboratory evolution (ALE) was used to generate an evolved 19–2 strain. Under 2-PE stress, its OD600 and growth rate increased by 86% and 22% than that of the parental strain, respectively. Through whole genome sequencing and reverse engineering, transcription factor Pdr1p mutation (C862R) was revealed as one of the main causes for increased 2-PE tolerance. Under 2-PE stress condition, Pdr1p mutation increased unsaturated fatty acid/saturated fatty acid ratio by 42%, and decreased cell membrane damage by 81%. Using STRING website, we identified Pdr1p interacted with some proteins, which were associated with intracellular ergosterol content, reactive oxygen species (ROS), and the ATP-binding cassette transporter. Also, the results of transcriptional analysis of genes encoded these proteins confirmed that Pdr1p mutation induced the expression of these genes. Compared with those of the reference strain, the ergosterol content of the PDR1_862 strain increased by 72%–101%, and the intracellular ROS concentration decreased by 38% under 2-PE stress. Furthermore, the Pdr1p mutation also increased the production of 2-PE (11% higher). Conclusions In the present work, we have demonstrated the use of ALE as a powerful tool to improve yeast tolerance to 2-PE. Based on the reverse engineering, transcriptional and physiological analysis, we concluded that Pdr1p mutation significantly enhanced the 2-PE tolerance of yeast by regulating the fatty acid proportion, intracellular ergosterol and ROS. It provides new insights on Pdr1p mediated 2-PE tolerance, which could help in the design of more robust yeasts for natural 2-PE synthesis.

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Adaptive responses of Kluyveromyces marxianus CCT 7735 to 2-phenylethanol stress: Alterations in membrane fatty-acid composition, ergosterol content, exopolysaccharide production and reduction in reactive oxygen species

Cited by 13 publications

References 50 publications

ROS Stress and Cell Membrane Disruption are the Main Antifungal Mechanisms of 2-Phenylethanol against Botrytis cinerea

ROS Stress and Cell Membrane Disruption are the Main Antifungal Mechanisms of 2-Phenylethanol against Botrytis cinerea

Spoilage Potential of Contaminating Yeast Species Kluyveromyces marxianus, Pichia kudriavzevii and Torulaspora delbrueckii during Cold Storage of Skyr

Evolutionary and reverse engineering in Saccharomyces cerevisiae reveals a Pdr1p mutation-dependent mechanism for 2-phenylethanol tolerance

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