Engineering of a Synthetic Metabolic Pathway for the Carbon-Conserving Production of Value-Added Compounds from Ethylene Glycol

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Due to growing environmental concerns, the development of biochemical processes for efficient utilization of carbon dioxide (CO2) for the production of value-added chemicals receives growing attention. Current technologies rely on (electro-)chemical reduction of CO2 to synthesis gas, formate or methanol, all of which can serve as substrates in microbial production processes. At present, methanol is the most popular C1-carbon substrate for use in fermentation processes. However, the implementation and optimization of methanol assimilation pathways in genetically amenable microorganisms is hampered by the fact that they overlap in large part with the natural metabolism of the host organisms. This makes it difficult to assure proper carbon flux repartitioning between regenerating a formaldehyde-accepting molecule, typically ribulose monophosphate or glycine, and diverting captured carbon towards the product. Addressing this problem, a synthetic metabolic pathway has recently been described which converts methanol to acetyl-CoA via the characteristic intermediate glycolaldehyde (GA). GA is directly produced from two molecules of formaldehyde, thus, elegantly by-passing the need of regenerating a formaldehyde-accepting molecule. With the present project proposal, we are putting forward the idea of extending the panel of synthetic metabolic pathways emanating from GA. Specifically, we propose a carbon-conserving synthetic metabolic pathway which converts GA into the four-carbon compound 2-oxo-4-hydroxybutyric acid from which several value-added products including threonine and 2,4-dihydroxybutyric acid can be derived. The synthetic pathway is composed of four enzymatic activities, out of which two have not been previously described and shall be engineered in this project. Although the proposed pathway is entirely compatible with the use of methanol, we shall demonstrate the function of the pathway using ethylene glycol (EG) as the substrate. The technological advantages of using EG instead of methanol are discussed in the project description.