GSK3 phosphorylates and activates trehalose-6-phosphate synthase to improve trehalose production and thermotolerance in Ganoderma lucidum

Glycogen synthase kinase 3 (GSK3) plays crucial roles in diverse organisms, yet its physiological functions in filamentous fungi remain poorly characterized. Here, we show that knockdown of GlGSK3 in Ganoderma lucidum leads to increased glycogen accumulation, reduced pyruvate and ATP production, and impaired hyphal growth and thermotolerance. GlGSK3 interacts with and phosphorylates trehalose-6-phosphate synthase (TPS) at Serine 529, enhancing its enzymatic activity. Consistent with the central role of TPS in trehalose biosynthesis, trehalose levels were significantly lower in gltps-kd and glgsk3-kd strains compared to the wild type. Similarly, gltps-kd strains also exhibited diminished hyphal growth and thermotolerance. Under heat stress, both GlGSK3 and GlTPS protein levels were upregulated, leading to increased GlTPS phosphorylation, enhanced enzymatic activity, and elevated trehalose accumulation. Together, these results uncover a key role for GlGSK3 and the GSK3-TPS module in G. lucidum in regulating growth and adaptation to environmental stress.