I. Untargeted metabolomics analysis of A. niger strain ES4 in the presence or absence of ethanol. This aim is to apply untargeted metabolomics platforms to globally measure levels of extracellular hydrophobic metabolites in A. niger cultures incubated with or without ethanol, in order to uncover metabolites potentially impacting resistance of A. niger toward ethanol.
II. Characterization of metabolites as metabolic responses to ethanol stress. In this aim, we propose to employ bioinformatics, chemical, analytical, and chromatographic techniques to characterize differential metabolites found in aim 1.
III. Identification of ethanol-incorporated metabolites. As A. niger strain ES4 was isolated from the outside wall of an ethanol tank, the strain might possess special metabolic or biotransformation pathways for making use of ethanol. We thus propose to trace the possible incorporation of ethanol into metabolites with increased levels in ethanol-treated samples found in aim 1 by using stable-isotope labelling mass spectrometry experiments with ethanol-d6.
IV. The correlation between metabolic responses and tolerance of A. niger toward ethanol. As ethanol tolerance of A. niger might correlate to its metabolic responses to ethanol. This aim is to investigate growth of A. niger ES4 in the presence of ethanol and the differential metabolites characterized in aim 2.
