Construction of an aldolase-dependent synthetic metabolic pathway for carbon-conserving bioproduct syntheses from ethylene glycol

• Applicant: Professor Dr.-Ing. Thomas Walther
• Subject Area: Biological Process Engineering
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565916835

Ethylene glycol (EG) is an attractive feedstock for industrial bioproduction that can be derived from CO2, plastic waste, or lignocellulosic biomass. While various bacterial species can grow on EG, natural pathways display a low carbon efficiency for acetyl-CoA production resulting in a 50 % loss of carbon in the form of CO2. Acetyl-CoA is a key metabolite in central metabolism serving as a precursor of biomass formation and/or biosyntheses of industrially-relevant products (e.g. isoprenoids, fatty acids). In recent years, various synthetic pathways have been designed for the carbon-conserving conversion of EG into acetyl-CoA. However, rates and/or affinity of key enzymes are too low to be physiologically relevant. In this proposal, these challenges are addressed by engineering a new, alternative carbon-conserving pathway for the synthesis of acetyl-CoA from EG composed of reactions all described to occur in natural enzymes. The new cyclic pathway relies on glycine as a glycolaldehyde-acceptor molecule, and it is regenerated during cleavage of threonine by an aldolase enzyme. The other product resulting from cleavage of threonine, acetaldehyde, is then converted into acetyl-CoA. An important enzyme in the pathway is 4-hydroxy threonine (4HT) deaminase, which deaminates 4HT into 2-oxo-4-hydroxybutyrate and has been shown to occur in the IlvA enzyme from Escherichia coli. However, since the enzyme is active on both threonine and 4HT, the implementation of the enzyme may divert carbon away from the cyclic pathway. Therefore, enzymes from other biological sources acting on substrates structurally related to 4HT (such as serine deaminase) will be investigated. Those will serve as the basis of a directed evolution approach that relies on random mutagenesis of encoding genes coupled with a high-throughput screening assay. Afterward, we shall employ a growth-based selection scheme to sequentially demonstrate the conversion of EG into acetyl-CoA in a tester strain auxotrophic for acetyl-CoA. Here, the model organism Pseudomonas putida has been chosen as the host for pathway implementation. The entire reaction sequence will be further transferred to a non-auxotrophic P. putida strain to demonstrate that EG indeed serves as a sole source of carbon and energy for biomass formation. Adaptive laboratory evolution shall be used as a means to improve pathway performance, and the work will be complemented with whole-genome sequencing analyses to ascertain genetic interventions responsible for improved performance. To demonstrate the potential of the new technology, we shall develop a production strain to show isoprenoid-precursor mevalonate from the synthetic pathway starting from EG. The engineered strains will be tested in bioreactor cultivations that will be accompanied by 13C labeling analyses. Overall, the project aims at the in vivo realization of a synthetic pathway for the bioconversion of EG as a significant contribution to the circular bioeconomy.

• DFG Programme: Research Grants