Engineering the robustness of Meyerozyma guilliermondii for efficient 2-phenylethanol biosynthesis

Microbial cell factories have enabled the sustainable synthesis of numerous chemicals, yet the production of cytotoxic compounds remains restricted by limited understanding of toxicity mechanisms and intrinsic tolerance. Here, by integrating non-rational genome evolution with systems metabolic engineering in Meyerozyma guilliermondii, we systematically dissected and rewired the cellular tolerance landscape under 2-phenylethanol (2-PE) stress. We uncovered previously uncharacterized endogenous tolerance elements that coordinately reallocate energy and reducing power, enhance glutathione-dependent redox buffering, and reinforce membrane and mitochondrial robustness. Functional reconstruction and mechanistic analyses establish design principles linking redox allocation, membrane physiology, and respiratory metabolism in 2-PE tolerance. Incorporation of these mechanistically defined modules enabled production of 7.09 g l^-1 2-PE without the use of in situ product recovery, demonstrating exceptional intrinsic tolerance. Further coupling this robust chassis with ISPR maximized the production to 13.43 g l^-1 2-PE during bioreactor cultivation. Together, this work provides a mechanistic and engineering framework for designing robust microbial platforms capable of producing inhibition-prone chemicals.