title: In vitro metabolic engineering for the production of α-ketoglutarate.
abstract: 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 10gL-1glucuronate with 92% yield of aKG within 5h. The maximum productivity of 2.8gL-1h-1is the second highest reported in the biotechnological synthesis of aKG.
authors.affiliation: Chair of Chemistry of Biogenic Resources, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Beer
authors.affiliation: Chair of Chemistry of Biogenic Resources, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Pick
authors.affiliation: Chair of Chemistry of Biogenic Resources, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany; Catalysis Research Center, Technical University of Munich, Ernst-Otto-Fischer-Str. 1, 85748 Garching, Germany. Electronic address: sieber@tum.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
mesh_terms: D053898:Biosynthetic Pathways / Q000502:physiology*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods; D020723:Glucuronic Acid / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000302:isolation & purification / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology; D014574:Uronic Acids / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D014574:Uronic Acids; D020723:Glucuronic Acid
keywords: Bubble reactor; Enzyme cascade reaction; In vitro reaction; Systems biocatalysis; α-ketoglutarate