Abstract
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.