PLS-SEM Reveals Growth-Mediated Regulatory Control of Withanolide Biosynthesis Under Micronutrient Stress in Withania coagulans (Stocks) Dunal

Withania coagulans (Stocks) Dunal is a medicinally important species valued for its bioactive steroidal lactones, particularly Withaferin A (WFA) and Withanolide A (WDA). Optimizing micronutrient regimes under in vitro conditions offers a promising strategy for enhancing secondary metabolite production; however, systematic comparative evaluation of essential and non-essential micronutrients across graded concentrations remains limited. The present study investigated the dose-dependent effects (1×–50× MS level) of Fe, Zn, Mn, Cu, and Co on withanolide accumulation. Cu exhibited the strongest elicitor effect, increasing withanolides up to ∼2-fold at 10× MS concentration, followed by decline at supra-optimal levels. Fe, Zn, and Mn displayed biphasic trends with defined optimal thresholds (20×–30× MS), producing significant enhancement relative to basal levels. In contrast, Co caused a consistent concentration-dependent suppression of both withanolides, with reductions exceeding 60% at higher doses. Principal Component Analysis (PCA) and hierarchical clustering (HCA) clearly separated treatments into growth-promoting and stress-dominated domains, with high-dose Co treatments clustering distinctly from essential micronutrients. Partial Least Squares Structural Equation Modeling (PLS-SEM) further revealed that secondary metabolite biosynthesis was mediated through integrated physiological pathways, with morphology emerging as the principal determinant of WFA (β ≈ 0.98) and WDA (β ≈ 0.66). The structural model demonstrated moderate-to-strong predictive accuracy (R 2 ≈ 0.47–0.51) for withanolides. These findings provide statistically validated, mechanistic insight into micronutrient-driven metabolic modulation and offer a robust framework for optimizing sustainable in vitro production of pharmacologically important withanolides in W. coagulans .