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Philipps-Universität Marburg
Abstract
In this thesis, Escherichia coli was engineered into growth-coupled biosensors with the capacity to detect a range of biologically and industrially relevant compounds with high specificity and sensitivity. Specifically, strains were developed to sense glycolaldehyde (GA) and glycerate by coupling their detection to essential metabolic processes through synthetic auxotrophies.
Two distinct GA biosensors were created: one based on pyridoxal-5-phosphate (PLP) auxotrophy and another on 2-ketoglutarate (2KG) auxotrophy. The PLP-based GA sensor was engineered by disrupting the canonical PLP biosynthesis pathway and redirecting it through an alternative GA-dependent route. In the 2KG-based sensor, the detection of GA was linked to a novel biosynthesis pathway of 2KG. Both types of sensor were subjected to extensive characterization and optimization to enhance GA detection, achieving an operational range that extends three orders of magnitude of GA concentration.
Furthermore, the GA biosensors were evaluated for in situ GA production through various metabolic pathways, including the oxidation of ethylene glycol, the Dahms and Weimberg pathways for xylose catabolism, demonstrating therefore their versatility and sensitivity. Moreover, new-to-nature enzymatic reactions for glycolate reduction were successfully implemented, highlighting the potential of these sensors for investigating synthetic metabolic networks in vivo.
In parallel, glycerate biosensors were developed by linking the synthesis of serine, an essential amino acid, to the presence of glycerate, providing an alternative to existing sensors that disrupt central carbon metabolism. The glycerate sensors presented here offer enhanced stability and accuracy in detecting glycerate. Additionally, a previously charactherized glycerate sensor was further engineered to improve its suitability for long-term cultivations. During this process, a novel function of the well-characterized succinate semialdehyde dehydrogenase enzyme was discovered.
The work presented here advances the fields of biotechnology and metabolic engineering by providing biosensors with the potential to serve as tools for the synthetic biology community.