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, and low enzyme levels can limit metabolic flux. Here, we used CRISPR interference (CRISPRi) to study the consequences of decreasing E. coli enzymes below wild-type levels. A pooled CRISPRi screen with 7,177 strains demonstrates that metabolism buffers fitness defects for hours after the induction of CRISPRi. We characterized the metabolome and proteome responses in 30 CRISPRi strains and elucidated three gene-specific buffering mechanisms: ornithine buffered the knockdown of carbamoyl phosphate synthetase (CarAB) by increasing CarAB activity, S-adenosylmethionine buffered the knockdown of homocysteine transmethylase (MetE) by de-repressing expression of the methionine pathway, and 6-phosphogluconate buffered the knockdown of 6-phosphogluconate dehydrogenase (Gnd) by activating a bypass. In total, this work demonstrates that CRISPRi screens can reveal global sources of metabolic robustness and identify local regulatory mechanisms that buffer decreases of specific enzymes. A record of this paper's transparent peer review process is included in the Supplemental Information.