Unraveling the proteome of Escherichia coli during L-phenylalanine production in a scale-down bioreactor.

Large-scale industrial bioprocesses are characterized by spatial and temporal inhomogeneities that expose microorganisms to fluctuating bioprocess conditions. These gradients can strongly influence cellular physiology, thereby affecting bioprocess robustness and product formation. Although phenotypic population heterogeneity under such conditions has been described, the underlying protein-level mechanisms remain poorly understood. In this study, the impact of environmental fluctuations on the proteome of an L-phenylalanine producing Escherichia coli strain was investigated. Samples were collected from cultivations in a well-mixed stirred-tank bioreactor and a two-compartment bioreactor operated at mean hydraulic residence times of τ = 103s and τ = 404s. Global proteome patterns were analyzed using principal component analysis (PCA), followed by differential protein abundance analysis and functional interpretation by gene ontology (GO) enrichment and clusters of orthologous groups (COG)-based analysis. PCA identified residence time in the bypass as the dominant factor shaping proteome variation. Prolonged exposure to limiting conditions at τ = 404s led to a distinct clustering of proteome profiles, clearly separated from both the stirred-tank bioreactor and the shorter residence time condition. GO enrichment analysis revealed increased abundance of proteins associated with stress response during the batch and product formation phases, and proteins related to cell communication during biomass production. Consistently, COG-based analysis showed a shift from growth- and production-associated proteins toward maintenance and stress adaptation in cells from the two-compartment bioreactor, with this shift becoming more pronounced at longer residence times.