Synergistic effects of exogenous inducers on ganoderic acid accumulation in Ganoderma lucidum: optimization and transcriptomic analysis

Ganoderic acids (GAs) are bioactive compounds in Ganoderma lucidum that have various pharmacological properties. Their biosynthesis is influenced by environmental factors and elicitors, but the synergistic effects of multiple inducers on GA accumulation are not well understood. This study examined the effects of calcium chloride (CaCl2), phenobarbital (PB), methyl jasmonate (MeJA), and 5-azacytidine (5-AC) as exogenous inducers. The results showed GA contents of 26.35 ± 0.67, 31.53 ± 0.30, 24.66 ± 0.82, and 26.61 ± 0.37 mg/g dry weight (DW), respectively. Using Box–Behnken design (BBD) based on the response surface methodology (RSM), the optimal combination of inducers was determined to be 400.00 μM CaCl2, 1,087.96 μM PB, 260.67 μM MeJA, and 823.04 μM 5-AC (the Mix treatment), which led to a GA content of 31.94 mg/g DW, a 47.67% increase compared with the untreated control. Under 125 μM MeJA and the Mix treatment, the contents of GA-B and GA-D increased by 121.50- and 68.94-fold, respectively. Transcriptomic analysis revealed 233, 216, 1858, 488, and 699 differentially expressed genes (DEGs) in the five treatment groups. Among them, 5, 5, 2, 6, and 5 upregulated mevalonic acid (MVA) pathway genes and 4, 7, 69, 10, and 36 Cytochrome P450 (CYP) DEGs were identified, including a common downregulated CYP gene, GLB03G000748, suggesting its role in negative regulation of GA biosynthesis. This study not only demonstrates an effective combined induction strategy for the enhanced production of GAs by G. lucidum but also, through transcriptomic analysis, provides crucial molecular insights into the regulatory mechanisms of GA biosynthesis.