Understanding and Engineering Metabolic Feedback Regulation of Amino Acid Metabolism in Escherichia coli

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. These chemical reactions comprise a large metabolic network that is subject to tight feedback-regulation of enzyme activities or abundances. However, even in intensively studied model organisms like Escherichia coli, the knowledge about the function of feedback-regulatory mechanisms and how they interact to control metabolism is still sparse. Therefore, the first goal of this study was to understand the function and relevance of metabolic feedback regulation using amino acid metabolism in E. coli as a case study. The second goal was to use the knowledge about metabolic feedback regulation to engineer microbial cell factories for the production of amino acids like L-arginine. In Chapter 1 we constructed a panel of 7 mutants with allosterically dysregulated amino acid pathways to uncover the relevance and function of allosteric feedback inhibition in vivo, which was so far only demonstrated by theoretical studies. By combining metabolomics, proteomics and flux profiling we could show that allosteric feedback inhibition is crucial to adjust a reserve of biosynthetic enzymes. Such enzyme overabundance originates from a sensitive interaction between control of enzyme activity (allosteric feedback inhibition) and enzyme abundance (transcriptional regulation). Furthermore, we used a metabolic model and CRISPR interference experiments to show that enzyme overabundance renders cells more robust against genetic perturbations. In Chapter 2 we increased fitness of a rationally engineered arginine overproduction strain by leaving a certain level of transcriptional regulation. Therefore, we titrated the transcription factor ArgR by CRISPRi and compared this different level of transcriptional regulation with an ArgR knockout strain. Using the CRISPRi approach we elevated the growth rates of an overproduction strain by two-fold compared to the knockout strain, without impairing arginine production rates and titer. Metabolomics and proteomics experiments revealed that slow growth of the knockout strain derives from limitations in pyrimidine nucleotide metabolism and that these limitations are caused by imbalances of enzyme level at critical branching points. Thus, we demonstrated the importance of balancing enzymes in an overproduction pathway and that CRISPRi is a suitable tool for this purpose. In Chapter 3 we show how cells respond to genetic perturbation on the molecular scale. Therefore, we perturbed amino acid biosynthesis genes with CRISPRi and analyzed the transcriptional response with GFP-reporter plasmids and proteomics. These experiments revealed that cells elevate the expression of genes in a perturbed pathway to counteract a genetic perturbation (We will refer to this mechanism as transcriptional compensation). Metabolomics and flow cytometry data of the wild-type and the allosteric mutant demonstrated the benefit of enzyme overabundance in response to genetic perturbations: Cells without overabundance showed a heterogenic transcriptional compensation even to mild perturbations, whereas in wild-type cells such mild perturbations were buffered by enzyme overabundance. In Chapter 4 we consider amino acid degradation pathways as an additional regulatory mechanism for the maintenance of end-product homeostasis Nutritional downshift experiments revealed increased robustness of allosteric mutants in which the respective degradation pathway was up-regulated. By dynamic metabolite measurements we showed that E. coli channels an excess of arginine into the degradation pathway. This overflow mechanism might be the reason for the robustness of allosteric mutants under dynamic conditions