Multi-omics analysis of CRISPRi-knockdowns identifies mechanisms that buffer decreases of enzymes in E. coli metabolism

Enzymes maintain metabolism and their concentration affects cellular fitness. High enzyme-levels are costly, but low enzyme-levels can limit metabolic flux. Here, we used CRISPR interference (CRISPRi) to study the consequences of decreasing metabolic enzymes in E. coli below wild-type levels. A time-resolved competition assay with a metabolism-wide CRISPRi library showed that fitness defects appeared late after induction of knockdowns. This suggested that metabolism is robust against decreases of enzymes. The metabolome and proteome of 30 CRISPRi strains revealed the mechanisms that enabled this robustness. First, substrates and allosteric effectors buffered knockdowns by increasing the activity of target-enzymes. Later, metabolite-transcription interactions compensated knockdowns by upregulating the target-pathway or bypass-pathways. For example, we found a new regulation strategy in which 6-phosphogluconate is responsible for bypassing bottlenecks in the pentose-p pathway via the Entner-Doudoroff-pathway. Thus, regulatory metabolites buffer decreases of enzyme-levels, which can occur in nature due to expression noise, mutations or environmental conditions.