Aspergillus niger upregulated glycerolipid metabolism and ethanol utilization pathway under ethanol stress

The knowledge of how Aspergillus niger responds to ethanol can lead to the design of strains with enhanced ethanol tolerance to be utilized in numerous industrial bioprocesses. However, the current understanding about the response mechanisms of A. niger toward ethanol stress remains quite limited. Here, we first applied a cell growth assay to test the ethanol tolerance of A. niger strain ES4, which was isolated from the wall near a chimney of an ethanol tank of a petroleum company, and found that it was capable of growing in 5% (v/v) ethanol to 30% of the ethanol-free control level. Subsequently, the metabolic responses of this strain toward ethanol were investigated using untargeted metabolomics, which revealed the elevated levels of triacylglycerol (TAG) in the extracellular components, and of diacylglycerol, TAG, and hydroxy-TAG in the intracellular components. Lastly, stable isotope labeling mass spectrometry with ethanol-d₆ showed altered isotopic patterns of molecular ions of lipids in the ethanol-d₆ samples, compared with the nonlabeled ethanol controls, suggesting the ability of A. niger ES4 to utilize ethanol as a carbon source. Together, the studies revealed the upregulation of glycerolipid metabolism and ethanol utilization pathway as novel response mechanisms of A. niger ES4 toward ethanol stress, thereby underlining the utility of untargeted metabolomics and the overall approaches as tools for elucidating new biological insights.