Abstract
The koji mold Aspergillus oryzae is widely adopted for producing rice wine, wherein koji mold saccharifies rice starch and sake yeast ferments glucose to ethanol. During rice wine brewing, the accumulating ethanol becomes a major source of stress for A. oryzae, and there is a decline in hydrolysis efficiency. However, the protective mechanisms of A. oryzae against ethanol stress are poorly understood. In the present study, we demonstrate that ethanol adversity caused a significant inhibition of mycelium growth and conidia formation in A. oryzae, and this suppressive effect increased with ethanol concentration. Transmission electron microscopy analysis revealed that ethanol uptake triggered internal cellular perturbations, such as irregular nuclei and the aggregation of scattered vacuoles in A. oryzae cells. Metabolic analysis uncovered an increase in fatty acid unsaturation under high ethanol conditions, in which a large proportion of stearic acid was converted into linoleic acid, and the expression of related fatty acid desaturases was activated. Our results therefore improve the understanding of ethanol adaptation mechanisms in A. oryzae and offer target genes for ethanol tolerance enhancement via genetic engineering.
Highlights
Aspergillus oryzae, commonly known as koji mold, is widely adopted for producing rice wine in East Asia [1] due to its outstanding capability of producing multienzymes such as α-amylase and glucoamylase [2]
During the process of saccharification, the starch in steamed rice is continuously hydrolyzed by α-amylase and glucoamylase provided by A. oryzae, and this process continues throughout the period of glucose production until the death of A. oryzae cells and the autolysis of mycelia [2]
The protective mechanism of A. oryzae against ethanol stress, which is critical to its survival and adaption to high-ethanol adversity in rice wine brewing, remains ambiguous
Summary
Aspergillus oryzae, commonly known as koji mold, is widely adopted for producing rice wine (sake, makgeolli, and huang-jiu) in East Asia [1] due to its outstanding capability of producing multienzymes such as α-amylase and glucoamylase [2]. The traditional alcoholic beverage of rice wine is produced through parallel fermentation, with a simultaneous saccharification of rice by the koji mold A. oryzae and ethanol fermentation by the sake yeast Saccharomyces cerevisiae [3]. It has been reported that the accumulation of ethanol affects the integrity of the cell membrane, inactivates cellular enzymes, and inhibits cell growth and viability, leading to cell death during fermentation [4,5]. High levels of ethanol are reported to destroy protein structure, leading to the denaturation of cellular proteins, such as the key glycolytic enzymes pyruvate kinase and hexokinase [6]. The ethanol tolerance mechanisms have been well documented in the budding yeast S. cerevisiae [7]. The protective mechanism of A. oryzae against ethanol stress, which is critical to its survival and adaption to high-ethanol adversity in rice wine brewing, remains ambiguous
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