Aims Cyanobacteria are prokaryotes performing oxygenic photosynthesis, and they can be engineered to harness solar energy for production of commodity and high-value chemicals by means of synthetic biology. The Cu2+-regulated petJ promoter (P-petJ), which controls the expression of the endogenous cytochrome c553, can be used for expression of foreign products in Synechocystis 6803. We aimed to disclose potential bottlenecks in application of the P-petJ in synthetic biology approaches. Methods and Results Quantitative label-free mass spectrometry revealed global proteome changes which occurred during nutrient conditions which repress or activate of P-petJ in Synechocystis 6803. Conclusions Some irreversible proteome alterations were discovered due to the copper stress, including downregulation of the ribosomal proteins, significant changes in protein amounts of the cell surface layer and the outer and inner membranes. Significance and Impact of the Study: This study revealed limitations in the use of P-petJ for biotechnological applications.