Trade-off Between Resistance and Persistence in High Cell Density Escherichia Coli Cultures

Microbes experience high cell density in many environments that come with diverse resource limitations and stresses. However, high density physiology remains poorly understood. We utilized well-controlled culturing systems to grow wild-type and metabolically engineered Escherichia coli strains into high cell densities (50–80 g Cdry cell weight L-1) and explore the associated transcriptional dynamics. Knowledge-enriched machine-learning-based analytics reveal distinct stress-related gene expression patterns that are consistent with a fundamental trade-off between resistance and persistence. We suggest that this trade-off explains observed growth arrests in high-density cultures and that it results from the disruption of cellular homeostasis, due to reallocation of limited cellular resources from resistance functions towards maintenance requirements of engineered production pathways. This study deepens our understanding of high-density physiology and demonstrates its importance to fundamental biomanufacturing challenges. Overall design: High cell density cultivation experiments were conducted following a common two-stage, batch-to-fed-batch, fermentation strategy in parallel sets of bioreactors. We studied five groups of E. coli strains with varying degree of engineering, including a wildtype BW25113 control strain (WT), different single gene knockout strains (SGKO), two tryptophan production strains (HMP3071 and empty plasmid control SDT551), and a plasmid-carrying melatonin production strain (HMP3427). After initiation of the fed-batch phase, growth was maintained with exponential feeding strategies, providing a continuous supply of media, O2 and pH buffering capacity to all strains.