The highest itaconic acid producing transformants, sCAD1 and sCADA, expressed the synthetic codon-optimized cadA gene and had the highest copy numbers at 11 and 6, respectively. The transformants sCAD7 sCAD8 and sCAD10, which produced between 4 and 8 mM itaconic acid after 14 days, had copy numbers of 2, 2 and 4, respectively. Although the sCAD10 strain had 4 copies of Ca it did not produce more than the strains with only 2 copies of cadA. In this particular case, the place of integration could negatively influence the level of expression compared to s and sCAD8. The sCAD6 strain, which did not produce any itaconic acid, also did not have a copy of the cadA gene.