Induced effect of Na+ on perylenequinones biosynthesis in submerged fermentation of endophytic fungus Shiraia sp. Slf14 via calcineurin signal transduction.

Metal ions critically regulate microbial secondary metabolism. This study investigated the regulatory roles of metal ions in Shiraia sp. Slf14, an endophytic fungus with exceptional perylenequinone (PQ) biosynthesis capacity. Among eight ions tested, Na⁺, K⁺, Mg²⁺, and high Ca²⁺ (1.0 g/L) markedly enhanced PQ yields, while Mn²⁺, Cu²⁺, Zn²⁺, and Fe³⁺ (≥ 0.5 g/L) exerted strong inhibition. Na⁺ emerged as the optimal elicitor, boosting production to 504.82 ± 8.62 mg/L (+ 57.96%) at 1.0 g/L without cytotoxicity. Na⁺ exhibited concentration- and time-dependent biphasic regulation. Critical innovation was achieved at 4 g/L Na⁺ added on day 4 (early-log phase), maximizing total PQ yield (1836.95 ± 45.21 mg/L, 5.75 × control), while concentrations > 12 g/L or late addition (≥ day 8) suppressed biosynthesis. Component-specific sensitivities were observed among five PQs. Furthermore, Na⁺ activated the calcineurin pathway: stress-induced Cam/Cna triggered calcineurin-mediated Crz1 nuclear translocation, upregulating key PQ genes (pksI, omef, hydroxylase). Concurrent induction of ena1 (Na⁺ exporter) and Ca²⁺-ATPase maintained ion homeostasis. In conclusion, our work pointed out monovalent cations (Na⁺/K⁺) as potent PQ biosynthetic stimulators in filamentous fungi and establish the Na⁺-calcineurin-PQ axis, offering a scalable strategy to overcome production bottlenecks in pharmaceutical PQ manufacturing.