Adaptive Laboratory Evolution for Enhanced Tyrosol Tolerance Enables High-Titer Salidroside Production in Escherichia coli

Salidroside, a valuable phenolic glycoside from Rhodiola rosea, holds significant pharmaceutical potential, yet its microbial synthesis is inhibited by the toxicity of the precursor tyrosol. Here, we address this bottleneck through adaptive laboratory evolution (ALE) of an Escherichia coli chassis, generating an evolved strain E40, with markedly enhanced tyrosol tolerance after 176 generations. Introducing a UDP-glycosyltransferase (AtUGT85A1) into E40 yielded strain SA40, which produced 6.3 g/L salidroside in shake flasks and achieved a final titer of 35.3 g/L in bioreactor fermentations with tyrosol feeding. Whole-genome sequencing and transcriptional analysis revealed that this enhanced tolerance is driven by a spatiotemporal synergy: the upregulation of the glutamate transporter GltS mitigates tyrosol-induced oxidative stress, while a robA mutation finely modulates the AcrAB efflux pump to control intracellular tyrosol availability. Importantly, this synergy establishes a precursor retention mechanism, maintaining high intracellular substrate concentrations necessary for efficient downstream glycosylation without compromising cell viability. This work establishes a robust and scalable platform for phenolic glycoside production and highlights the critical importance of precursor retention mechanisms in resolving the tolerance-yield trade-off when engineering microbial cell factories for toxic intermediates.