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A metabolism-wide CRISPRi library expands the measurable E. coli metabolome - LC-MS2 12C/13C dataset

ABSTRACT: In this study, we developed a workflow to systematically and selectively induce increases in metabolites by knocking down enzymes with CRISPR interference (CRISPRi). Therefore, we created a sorted CRISPRi library targeting all 1,515 metabolic genes in the most recent genome-scale metabolic model of E. coli (iML15159). In a first step, we screened the metabolome of the CRISPRi library with a fast flow-injection mass spectrometry, which revealed strong and specific accumulation of 36% of the predicted metabolites in the iML1515 model. The accumulating metabolites were unique to certain knockdowns, especially those metabolites associated with the CRISPRi targeted-pathway. Therefore, we followed-up on accumulating metabolites that were directly associated to the CRISPRi target-gene (i.e. all reactants of the respective enzyme) and measured their fragmentation spectra and chromatographic retention times with LC-MS2. This resulted in experimental fragmentation spectra and chromatographic retention times of 111 metabolites. Three of these fragmentation spectra were verified by 13C labelling experiments.

INSTRUMENT(S): 6546 Q-TOF LC/MS (Agilent instrument model)

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Prof. Hannes Link

PROVIDER: MSV000095538 | MassIVE | Wed Aug 07 05:52:00 BST 2024

REPOSITORIES: MassIVE

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A metabolism-wide CRISPRi library expands the measurable E. coli metabolome - LC-MS2 dataset

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