The metabolome of an E. coli CRISPRi library identifies benefits of minimal metabolite levels and targets for engineering

Metabolite concentration changes can have broad consequences on the function and robustness of metabolic networks. Here, we measured the metabolome response of 1,515 CRISPR interference (CRISPRi) E. coli strains targeting all genes in the i ML1515 metabolic model. Metabolites that are hardly measurable in wild-type E. coli accumulated in specific CRISPRi strains, indicating that they are normally maintained at low levels. We confirmed metabolite accumulation using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated putative reference spectra for 102 metabolites for which no MS 2 data had previously been available. We show that minimal metabolite levels are beneficial because they (1) enable substrate level regulation of enzyme activity, (2) prevent competitive inhibition, and (3) suppress side reactions. However, minimal metabolite pools also limit flux through engineered pathways. For example, low levels of farnesyl diphosphate (frdp) constrained a synthetic carotenoid pathway, and we show that the knockdown of octaprenyl diphosphate synthase (IspB) increased frdp levels and carotenoid production. A record of this paper's transparent peer review process is included in the supplemental information.