In vitro metabolic engineering for the production of α-ketoglutarate

•In vitro synthetic enzyme cascade towards α-ketoglutarate from renewable resource.•92 % yield of α-ketoglutarate and productivity of 2.8 g L−1 h−1.•Cofactor supply is crucial for a successful process and can be achieved in two ways.•Use of a bubble reactor for oxygen saturation accelerates the cascade reaction from 150 h to 5 h.•Major obstacle remaining is enzyme stability at oxygen saturation.

α-Ketoglutarate (aKG) represents a central intermediate of cell metabolism. It is used for medical treatments and as a chemical building block. Enzymatic cascade reactions have the potential to sustainably synthesize this natural product. Here we report a systems biocatalysis approach for an in vitro reaction set-up to produce aKG from glucuronate using the oxidative pathway of uronic acids. Because of two dehydrations, a decarboxylation, and reaction conditions favoring oxidation, the pathway is driven thermodynamically towards complete product formation. The five enzymes (including one for cofactor recycling) were first investigated individually to define optimal reaction conditions for the cascade reaction. Then, the kinetic parameters were determined under these conditions and the inhibitory effects of substrate, intermediates, and product were evaluated. As cofactor supply is critical for the cascade reaction, various set-ups were tested: increasing concentrations of the recycling enzyme, different initial NAD+ concentrations, as well as the use of a bubble reactor for faster oxygen diffusion. Finally, we were able to convert 10 g L−1 glucuronate with 92% yield of aKG within 5 h. The maximum productivity of 2.8 g L−1 h−1 is the second highest reported in the biotechnological synthesis of aKG.