AOX Affects the Synthesis of Polysaccharides by Regulating the Reactive Oxygen Species in Ganoderma lucidum

Alternative oxidase (AOX) is a terminal oxidase in the mitochondrial electron transport chain that does not contribute to the generation of ATP. It plays a critical role in maintaining the balance between reactive oxygen species (ROS) production and intracellular redox homeostasis within the mitochondria. In the study, overexpression and knockdown approaches were employed to investigate the function of AOX. AOX-silenced strains (AOXi3 and AOXi25) and AOX-overexpressed strains (OE-AOX2 and OE-AOX21) were constructed. The ROS level and transcription level of the antioxidant-system-related genes, including phosphoglucomutase (pgm) and phosphomannose isomerase (pmi), were differentially upregulated in silenced strains, whereas the opposite effect was observed in the AOX-overexpressed strains. Compared with the wild type (WT), the polysaccharide production of AOXi25 was significantly increased by approximately 38%, while OE-AOX21 was significantly decreased by 80%. Six extracellular polysaccharides (EPSs) were extracted and purified from the WT, OE-AOX21, and AOXi25 strains. These EPSs, consisting of both neutral and acidic polysaccharides, were composed of five different monosaccharides in varying proportions. The average relative molecular masses were 1.68 × 10³, 2.66 × 10³, 1.67 × 10³, 2.42 × 10³, 1.12 × 10³, and 2.35 × 10³ kDa, respectively. Antioxidant assays demonstrated that all EPSs exhibited strong free radical scavenging activity with the acidic polysaccharide from AOXi25 showing the highest efficiency in ABTS⁺ scavenging. These findings highlight the significant role of AOX-derived ROS in regulating polysaccharide synthesis and accumulation in Ganoderma lucidum.