Timescale-dependent metabolic adaptation governs dynamic Crabtree regulation in Saccharomyces cerevisiae

Abstract

The Crabtree effect in Saccharomyces cerevisiae involves rapid metabolic switching between respiratory and fermentative states under dynamic conditions, yet its regulatory principles across different perturbation timescales remain poorly understood. Here, we imposed short-term glucose pulses and long-term dilution-rate perturbations on steady-state yeast cultures with extracellular physiological analysis, time-resolved intra- and extracellular metabolomics, proteomics, and constraint-based flux analysis to investigate dynamic Crabtree regulation. During short-term glucose pulses, ethanol accumulation scaled linearly with perturbation intensity, while approximately 33% of the incoming glucose carbon was consistently redirected to fermentation, revealing a conserved carbon-partitioning strategy. Under long-term dilution rate perturbations, abrupt shift-up triggered rapid overflow metabolism, respiratory imbalance, and growth suppression, whereas gradual ramp-up enabled coordinated respiratory adaptation and delayed fermentation onset. These results demonstrate that ethanol overflow does not require complete respiratory inhibition, but instead arises from a timescale mismatch between rapid glycolytic activation and slower mitochondrial adaptation. Furthermore, metabolite profiling identified fructose−1,6-bisphosphate (FBP), trehalose metabolism, and the glutamate/glutamine (Glu/Gln) ratio as key regulatory nodes coordinating carbon flux, nitrogen assimilation, energy metabolism, and phosphate homeostasis. Together, these findings establish a hierarchical and timescale-dependent framework for understanding dynamic Crabtree regulation and provide insights for engineering robust yeast cell factories under fluctuating industrial environments.

Keywords

Saccharomyces cerevisiae / Crabtree effect / Time-resolved metabolomics / Glucose pulse / Accelerostat

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Funding

National Key Research and Development Program of China(2024YFA0920300)

National Natural Science Foundation of China(22578122)

Natural Science Foundation of Shanghai Municipality(25ZR1402110)

Taishan Scholars Program of Shandong Province(tspn202408281)

the Explorers Program of Shanghai (Basic Research Funding)(25TS1401100)

the Jiangsu Provincial Major Science and Technology Special Project(BG2025044)

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Jiangnan University