Search

Escherichia coli, a common microbial host for industrial bioproduction, experiences a highly dynamic environment in industrial-scale bioreactors due to significant glucose and oxygen gradients. In this study, we mimic the combined gradients of glucose and oxygen in high-throughput bioreactors to study the transcriptional response of E. coli to industrial-scale conditions. Under oscillating oxygen conditions, E. coli formed less biomass and accumulated the anaerobic by-product acetate. With respect to oxygen-responsive genes, we found that genes of the TCA cycle and of different electron transport chain complexes were differentially expressed. A global analysis of the expression data revealed that oxygen oscillations had caused a transition towards a catabolite-repressed state and upregulation of several stress-related regulatory programs. Interestingly, the transcriptional changes persisted after oxygen limitation stopped. In contrast, the changes we observed due to glucose starvation, such as induction of the stringent response, were primarily transient. Most importantly, we found that effects of combined oxygen and glucose oscillations were distinct from the ones of oxygen and substrate oscillations alone, suggesting an important interplay between the different metabolic regimes in industrial-scale bioreactors., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Engineering microbial cells for the commercial production of biomolecules and biochemicals requires understanding how cells respond to dynamically changing substrate (feast-famine) conditions in industrial-scale bioreactors. Scale-down methods that oscillate substrate are commonly applied to predict the industrial-scale behavior of microbes. We followed a compartment modeling approach to design a scale-down method based on the simulation of an industrial-scale bioreactor. This study uses high cell-density scale-down experiments to investigate Escherichia coli knockout strains of five major glucose-sensitive transcription factors (Cra, Crp, FliA, PrpR, and RpoS) to study their regulatory role during glucose oscillations. RNA-sequencing analysis revealed that the glucose oscillations caused the down-regulation of several stress-related functions in E. coli . An in-depth analysis of strain physiology and transcriptome revealed a distinct phenotype of the strains tested under glucose oscillations. Specifically, the knockout strains of Cra, Crp, and RpoS resulted in a more sensitive transcriptional response than the control strain, while the knockouts of FliA and PrpR responded less severely. These findings imply that the regulation orchestrated by Cra, Crp, and RpoS may be essential for robust E. coli production strains. In contrast, the regulation by FliA and PrpR may be undesirable for temporal oscillations in glucose availability., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)