Itaconic acid is a value-added organic acid that is widely applied in industrial production. It can be converted from citric acid by some microorganisms including Aspergillus terreus and Aspergillus niger. Because of high citric acid production (more than 200 g/L), A. niger strains may be developed into powerful itaconic acid-producing microbial cell factories. In this study, industrial citric acid-producing strain A. niger YX-1217, capable of producing 180.0-200.0 g/L, was modified to produce itaconic acid by metabolic engineering. A key gene cadA encoding aconitase was expressed in A. niger YX-1217 under the control of three different promoters. Analyses showed that the PglaA promoter resulted in higher levels of gene expression than the PpkiA and PgpdA promoters. Moreover, the synthesis pathway of itaconic acid was extended by introducing the acoA gene, and the cadA gene, encoding aconitate decarboxylase, into A. niger YX-1217 under the function of the two rigid short-peptide linkers L1 or L2. The resulting recombinant strains L-1 and L-2 were induced to produce itaconic acid in fed-batch fermentations under three-stage control of agitation speed. After fermentation for 104 h, itaconic acid concentrations in the recombinant strain L-2 culture reached 7.2 g/L, which represented a 71.4% increase in itaconic acid concentration compared with strain Z-17 that only expresses cadA. Therefore, co-expression of acoA and cadA resulted in an extension of the citric acid metabolic pathway to the itaconic acid metabolic pathway, thereby increasing the production of itaconic acid by A. niger.,© King Abdulaziz City for Science and Technology 2020.