Overexpression of a C4-dicarboxylate transporter is the key for rerouting citric acid to C4-dicarboxylic acid production in Aspergillus carbonarius

Lei Yang, Eleni Christakou, Jesper Vang, Mette Lübeck, Peter Stephensen Lübeck

C4-dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. Previous studies on organic acid production by Aspergillus carbonarius, which is capable of producing high amounts of citric acid from varieties carbon sources, have revealed its potential as a fungal cell factory. Earlier attempts to reroute citric acid production into C4-dicarboxylic acids have been with limited success. In this study, a glucose oxidase deficient strain of A. carbonarius was used as the parental strain to overexpress a native C4-dicarboxylate transporter and the gene frd encoding fumarate reductase from Trypanosoma brucei individually and in combination. Impacts of the introduced genetic modifications on organic acid production were investigated in a defined medium and in a hydrolysate of wheat straw containing high concentrations of glucose and xylose. In the defined medium, overexpression of the C4-dicarboxylate transporter alone and in combination with the frd gene significantly increased the production of C4-dicarboxylic acids and reduced the accumulation of citric acid, whereas expression of the frd gene alone did not result in any significant change of organic acid production profile. In the wheat straw hydrolysate after 9 days of cultivation, similar results were obtained as in the defined medium. High amounts of malic acid and succinic acid were produced by the same strains. This study demonstrates that the key to change the citric acid production into production of C4-dicarboxylic acids in A. carbonarius is the C4-dicarboxylate transporter. Furthermore it shows that the C4-dicarboxylic acid production by A. carbonarius can be further increased via metabolic engineering and also shows the potential of A. carbonarius to utilize lignocellulosic biomass as substrates for C4-dicarboxylic acid production.