Itaconic acid (IA) has many versatile applications in science and medicine. Engineering Escherichia coli can provide a reliable route for IA production, where cis -aconitate decarboxylase (CAD) is one essential enzyme in the process. The synthetic cad gene has been cloned into various vectors to improve the expression rate and folding activities. The strain bearing plasmid pTrc99a- cad displays a significant amount of IA production, while the CAD protein expressed from the same plasmid displays the highest enzymatic activity in vitro. To improve the reliability of forming the cis -aconitate intermediate, the aconitase genes from Aspergillus terreus have been cloned into the pTrc99a vector. The expression of aconitate hydratase leads to a peak at 10.3 min in the HPLC spectra after 4 hours incubation. The peak will be analyzed to determine whether it is cis -aconitate. The obtained data will provide data on possible metabolic pathways of IA production in Aspergillus terreus.