Here, we described a novel strategy for the production of itaconic acid in Escherichia coli by self-assembly of aconitase (ACO) and cis-aconitate decarboxylase (CAD) existing in the metabolic pathway of itaconic acid via the protein-peptide interactions of PDZ domain and PDZ ligand. Co-expression of ACO and CAD in E. coli (uCA) resulted in low levels of itaconate (117.25mg/L) after 48h fermentation while the itaconate titre was significantly improved up to 222.15mg/L by self-assembly of ACO-PDZ (APd) and CAD-PDZlig (CPl) in E. coli (sPP) under the same conditions. To further confirm the effect of self-assembly, the itaconate catalyzed from sodium citrate was determined. The sPP was extra efficacious in the early catalytic period, showing approximately threefold itaconate yields increased after 2h catalysis, when compared to uCA. Furthermore, the itaconate production of sPP was increased from 5 to 8.7g/L after 30h of reaction compared to uCA. This self-assembly strategy showed remarkable potential for the further improvement of itaconate production. Biotechnol. Bioeng. 2017;114: 457-462. (c) 2016 Wiley Periodicals, Inc.