LAUSR

Summary Targeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.