Metabolic Engineering of Escherichia coli for Overproduction of Alpha-Ketoglutarate Using Crude Glycerol

Alpha-ketoglutarate (alpha-KG) is a multifunctional chemical with significant and wide applications in industry. This study developed a sustainable production of alpha-KG based on crude glycerol by reprogramming Escherichia coli. The metabolic pathways leading to the synthesis of byproducts were blocked, and the anaplerotic route was manipulated to direct the glycolytic flux into the tricarboxylic acid (TCA) cycle. The strain was further engineered by recruiting the alpha-KG-insensitive gltA and removing mdh to render the oxidative TCA cycle incomplete. As a result, the engineered strain enabled the overproduction of alpha-KG. The microbial fermentation was then conducted in a fed-batch mode. By adjusting the glycolytic flux, the producer strain finally produced alpha-KG with the titer, productivity, and conversion yield on crude glycerol reaching 44 g/L, 1.58 g/L/h, and 0.51 g/g, respectively. Overall, the results indicate that the developed E. coli shows promise for producing alpha-KG from crude glycerol.