Assessment of ethanol tolerance of Kluyveromyces marxianus CCT 7735 selected by adaptive laboratory evolution.

Kluyveromyces marxianus CCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerant K. marxianus CCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26 g/g/h) higher than those presented by the ETS1 (0.22 g/g/h), ETS2 (0.17 g/g/h), and ETS3 (0.17 g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4. KEY POINTS: • K. marxianus ethanol-tolerant strains were selected by adaptive laboratory evolution. • Valine and metabolites of the TCA cycle were accumulated in the ETS4. • High contents of fatty acids and ergosterol contributed to enhance ethanol tolerance.

Mass Spectrometry-Based Metabolomics to Investigate the Effect of Mechanical Shaking on Sauvignon Blanc Berry Metabolism.

Previous commercial studies carried out in New Zealand showed that mechanical shaking significantly reduced the incidence of Botrytis cinerea infection in wine grapes. However, the reasons behind this reduction are not well understood. Here, we employed a metabolomics approach to gain insights into the biochemical changes that occur in grape berries due to mechanical shaking. Berry samples were analyzed using three different analytical approaches including gas chromatography and mass spectrometry (MS), liquid chromatography and MS, and imaging mass spectrometry (IMS). Combined data provided a comprehensive overview of metabolic changes in grape berry, indicating the initiation of different stress mitigation strategies to overcome the effect of mechanical shaking. Berry primary metabolism was distinctly altered in the green berries in response to mechanical shaking, while secondary metabolism significantly changed in berries collected after veraison. Pathway analysis showed upregulation of metabolites related to nitrogen and lipid metabolism in the berries from shaken vines when compared with controls. From IMS data, we observed an accumulation of different groups of metabolites including phenolic compounds and amino and fatty acids in the areas near to the skin of berries from shaken vines. This observation suggests that mechanical shaking caused an accumulation of these metabolites, which may be associated with the formation of a protective barrier, leading to the reduction in B. cinerea infection in berries from mechanically shaken vines.