How to Characterize and Exploit Intracellular Transport Processes By Metabolic Engineering

The in vitro characterization of transmembrane carrier proteins is challenging, because it normally requires the overexpression and reconstitution into liposomal systems. Here, we present a new strategy making use of an in vivo metabolic engineering approach to describe the substrate specificity of a mitochondrial carrier namely MttA of Aspergillus terreus. This mitochondrial carrier protein is involved in the biosynthesis of itaconic acid. We analyzed the transport specificity of MttA making use of different metabolically engineered Aspergillus niger strains. It was found that MttA preferentially transports cis-aconitic acid over citric acid and does not transport itaconic acid. The expression of MttA in selected A. niger strains results in secretion of aconitic acid (up to 9 g/L). Furthermore, the mitochondrial localization of this protein is confirmed using fluorescence microscopy. MttA can be used in further strain engineering strategies to transport cis-aconitic acid from the mitochondria to the cytosol for the production of itaconic acid or related metabolites.

Thus, metabolic engineering can be used for both the in vivo characterization of transport protein functions like MttA and to make use of this protein by creating aconitic acid producing strains.