Expression pattern, subcellular localization of <i>Aspergillus oryzae</i> ergosterol synthases, and their effects on ergosterol and fatty acid metabolism

Ergosterol is an important component of fungal cell membranes and participates in the regulation of its fluidity, permeability, and material transport. In our study, we found that the ergosterol biosynthesis pathway in <i>Aspergillus oryzae</i> is more complex than that in <i>Saccharomyces cerevisiae</i>, involving 49 enzymes and 25 reactions. In this study, we systematically analyzed the expression pattern and subcellular localization of <i>A. oryzae</i> ergosterol synthases and their effects on ergosterol and fatty acid metabolism. The results showed that ergosterol synthase genes were distributed across the eight chromosomes of the <i>A. oryzae</i> genome and were differentially expressed at different growth times. Subcellular localization analysis revealed that the mevalonate biosynthesis-related enzymes were localized in the cytoplasm, mitochondria, and peroxisomes; farnesyl pyrophosphate biosynthesis-related enzymes were mainly localized in the cytoplasm, peroxisomes, and mitochondria; while several ergosterol biosynthesis-related enzymes were localized in the endoplasmic reticulum and lipid droplets. Overexpression (OE) of these enzymes affected both ergosterol and fatty acid contents in <i>A. oryzae</i>. The ergosterol content was the highest in the <i>AoHmgB</i>-OE strain and the lowest in the <i>AoHmgA</i>-OE strain, while the fatty acid content was the highest in the <i>AoErg26B</i>-OE strain and the lowest in the <i>AoErg26A</i>-OE strain. Moreover, the ergosterol content was significantly increased in the <i>AoHmgB</i>/<i>AoErg7B</i>-, <i>AoHmgB</i>/<i>AoErg9</i>-, and <i>AoErg1</i>/<i>AoErg7B</i>-co-OE strains compared to the single gene OE strains. Our study results may offer a scientific foundation for genetic engineering research focusing on lipid metabolism in <i>A. oryzae</i> and related fungi, thereby facilitating the creation of fungal strains with enhanced ergosterol production.IMPORTANCEErgosterol, an important fungal cell membrane component, participates in the regulation of membrane fluidity, permeability, and material transport. Previous studies have demonstrated that the ergosterol biosynthesis pathway in <i>Aspergillus oryzae</i> exhibits greater complexity compared to that in <i>Saccharomyces cerevisiae</i>; nonetheless, research on the ergosterol biosynthesis pathway in <i>A. oryzae</i> remains limited. In this study, we determined the expression pattern and subcellular localization of ergosterol biosynthesis-related enzymes in <i>A. oryzae</i>. Additionally, we assessed the effects of the overexpression (OE) of ergosterol biosynthesis-related genes on ergosterol and fatty acid contents in <i>A. oryzae</i>. Therefore, our study may provide a scientific basis for genetic engineering research on lipid metabolism in <i>A. oryzae</i> and other fungal species.