Rhodotorula mucilaginosa shows adaption to a broad range of Pb²⁺ stress. In this study, three key pathways, i.e., glycolysis (EMP), the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), were investigated under 0-2,500 mg · L^-1 Pb stress, primarily based on biochemical analysis and RNA sequencing. R. mucilaginosa cells showed similar metabolic response to low/medium (500/1000 mg · L^-1) Pb²⁺ stress. High (2,500 mg · L^-1) Pb²⁺ stress exerted severe cytotoxicity to R. mucilaginosa. The downregulation of HK under low-medium Pb²⁺ suggested a correlation with the low hexokinase enzymatic activity in vivo. However, IDH3, regulating a key step of circulation in TCA, was upregulated to promote ATP feedstock for downstream OXPHOS. Then, through activation of complex I & IV in the electron transport chain (ETC) and ATP synthase, ATP production was finally enhanced. This mechanism enabled fungal cells to compensate for ATP consumption under low-medium Pb²⁺ toxicity. Hence, R. mucilaginosa tolerance to such a broad range of Pb²⁺ concentrations can be attributed to energy adaption. In contrast, high Pb²⁺ stress caused ATP deficiency. Then, the subsequent degradation of intracellular defense systems further intensified Pb toxicity. This study correlated responses of EMP, TCA, and OXPHOS pathways in R. mucilaginosa under Pb stress, hence providing new insights into the fungal resistance to heavy metal stress. IMPORTANCE Glycolysis (EMP), the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS) are critical metabolism pathways for microorganisms to obtain energy during the resistance to heavy metal (HM) stress. However, these pathways at the genetic level have not been elucidated to evaluate their cytoprotective functions for Rhodotorula mucilaginosa under Pb stress. In this study, we investigated these three pathways based on biochemical analysis and RNA sequencing. Under low-medium (500-1,000 mg · L^-1) Pb²⁺ stress, ATP production was stimulated mainly due to the upregulation of genes associated with the TCA cycle and the electron transport chain (ETC). Such an energy compensatory mechanism could allow R. mucilaginosa acclimation to a broad range of Pb²⁺ concentrations (up to 1000 mg · L^-1). In contrast, high (2500 mg · L^-1) Pb²⁺ stress exerted its excessive toxicity by provoking ATP deficiency and damage to intracellular resistance systems. This study provided new insights into R. mucilaginosa resistance to HM stress from the perspective of metabolism.

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