The Role of ATG8 in Promoting Lipid Accumulation in the Oleaginous Fungus Mucor circinelloides During Nitrogen Limitation

Autophagy is a central cellular process that recycles intracellular components and supplies precursors for biosynthesis. As a key regulator of autophagosome formation, autophagy-related protein 8 (ATG8) plays an essential role in macromolecular degradation and in the availability of lipid precursors. However, whether enhanced autophagic flux promotes lipid accumulation in oleaginous fungi remains unclear. In this study, atg8-1 and atg8-2 were homologously overexpressed in the oleaginous fungus Mucor circinelloides to evaluate their roles in lipid biosynthesis. The engineered strains McATG8-1T2 and McATG8-2T2 showed significantly increased total fatty acid (TFA) contents (32.9% and 32.5%), representing improvements of 15.0% and 13.7% compared with the control. γ-Linolenic acid levels were also elevated to 16.9% and 16.5%, relative increases of 25.2% and 22.0%, respectively. RT-qPCR analysis revealed coordinated upregulation of genes involved in autophagy, central carbon metabolism, lipid biosynthesis, and the pentose phosphate pathway. Ethanolamine supplementation further enhanced lipid accumulation, increasing TFA contents by 12.2–14.6%. In addition, inhibition of target of rapamycin complex 1 using rapamycin produced a strong synergistic effect with atg8 overexpression, leading to substantial lipid increases under nitrogen-limited and nitrogen-rich conditions. Collectively, these findings demonstrated that ATG8-mediated autophagy enhanced lipid accumulation and acted as a key determinant of lipid synthesis flux.