Abstract:
Metabolism is the core of what we consider to be a living cell. It covers all chemical reactions that are necessary to break down nutrients and convert them into energy and cellular building blocks for growth. Escherichia coli is arguably the most studied model organism. Traits that make E. coli especially suitable for genetic engineering and convenient to study bacterial metabolism include: rapid growth, high yields, cost effectiveness, genetic accessibility and robust handling. To study metabolism, metabolomics serves as a valuable tool within the field of systems biology, allowing researchers to explore concentration changes of metabolites and metabolic fluxes. Here, I show in four structured chapters recent advances in mass spectrometry-based metabolomics to engineer and investigate E. coli central metabolism on a systems-level.
I explored innovative approaches in metabolic engineering and metabolomics to advance our understanding of bacterial metabolism, using the model organism E. coli. I introduced a novel protein-based barcode system for CRISPR interference strains, enabling real-time tracking and phenotyping while avoiding the need for sequencing sgRNAs. This method offers a cost-effective solution for simultaneous tracking and phenotyping of CRISPRi Libraries. Additionally, I developed a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approach to quantify sugar phosphate isomers in E. coli, providing insights into the central metabolism. By perturbing metabolic pathways through CRISPR interference, I revealed dynamic changes in hexose and pentose phosphates, underscoring the potential of this method for various organisms. Further, I investigated the metabolic adaptations of E. coli during its transition to chemoautotrophy, shedding light on the roles of autotrophic-enabling mutations in energy and carbon metabolism. This has implications for the conversion of CO2 into valuable compounds in sustainable bioprocesses. Finally, I introduced a temperature-controlled two-stage process to produce arginine in E. coli, demonstrating the decoupling of growth and overproduction using a temperature switch. This strategy holds promise for microbial production of amino acids and valuable compounds.
Together, this work contributes to our understanding of cellular metabolism, offering innovative tools and methodologies for metabolic engineering, metabolomics, and the sustainable production of valuable compounds.
Metabolic engineering