Combining Metabolomics and CRISPRi to Understand and Engineer Bacterial Metabolism
We used high-throughput flow-injection mass spectrometry to measure metabolites in thousands of samples and systematically map metabolome changes in CRISPRi E. coli strains targeting all genes in a genome-scale metabolic model. Many metabolites that are barely detectable in wild-type cells accumulated in specific knockdowns, indicating that cells actively maintain them at minimal levels.
Using LC/MS/MS, we validated these findings and generated reference spectra for more than 100 previously uncharacterized metabolites.
Our data show that low metabolite concentrations support metabolic robustness by enabling substrate-level regulation, limiting competitive inhibition, and suppressing side reactions. At the same time, minimal metabolite pools can restrict flux through engineered pathways.
We will show examples of how metabolic bottlenecks can be used to redirect metabolic flux into engineered pathways.
Presenter: Anna Walke (LC/MS Application Engineer, Agilent Technologies)
Anna joined Agilent as an application specialist for LC/MS focusing on small molecules in life science and metabolomics applications. Previously, she developed LC-MS based methods for clinical biomarker studies during her PhD in analytical chemistry and supported several fluorescence microscopy studies. Her background involves analytical chemistry, biochemistry, and food chemistry.
Presenter: Prof. Dr. Hannes Link (Professor at the University of Tübingen, Director of the Interfaculty Institute for Microbiology and Infection Medicine)
Prof. Dr. Hannes Link is a W3 Professor at the University of Tübingen and Director of the Interfaculty Institute for Microbiology and Infection Medicine. He studied Chemical Engineering at the Technical University of Munich and completed his PhD in Biochemical Engineering there. He subsequently conducted postdoctoral research at the Institute of Molecular Systems Biology at ETH Zurich and led an independent research group at the Max Planck Institute for Terrestrial Microbiology in Marburg before being appointed to Tübingen in 2020. His research integrates systems biology, synthetic biology, and metabolic engineering. The focus is on quantitative analysis and rational control of bacterial metabolism. His group applies multi-omics approaches, particularly quantitative metabolomics to uncover regulatory principles in Escherichia coli and to use these insights for biotechnology.
