CRISPRi-knockdowns of enzymes identify mechanisms that enable metabolic robustness in E. coli

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 7177 strains suggested that metabolism is robust against knockdowns, because fitness defects appeared several hours after induction of CRISPRi. Metabolome and proteome responses in 30 CRISPRi strains were local and specific. For 3 CRISPRi strains, we show that metabolome and proteome responses can buffer the knockdown. 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. 6-phosphogluconate buffered the knockdown of 6-phosphogluconate dehydrogenase(Gnd) by activating a bypass. Thus, CRISPRi enforces local and specific responses inthe metabolic network, which reveal regulatory mechanisms that buffer decreases of enzymes.