Penicillin acylase (PAC) from Escherichia coli has a complex enzyme formation mechanism that is unusual for prokaryotic proteins. In this study, PAC is used as a model protein to demonstrate an important concept of developing genetic strategies for improving recombinant protein production in E. coli; namely, the bottleneck gene expression step(s) limiting recombinant protein production must be precisely identified. Using the strong promoter system of trc for regulation of pac gene expression, the overproduction of PAC was often limited by translocation and/or periplasmic processing steps, resulting in intracellular accumulation of various types of PAC precursors. The over-accumulated PAC precursors formed inclusion bodies in the cytoplasm and/or periplasm. The periplasmic protease DegP could efficiently assist periplasmic processing of PAC precursors and, therefore, the amount of periplasmic inclusion bodies was significantly reduced. The PAC inclusion bodies remaining in DegP-coexpressing cells were located in the cytoplasm where the DegP function could not reach. These results indicate that the activity of DegP expression based on pKS12 was high enough for processing the over-accumulated periplasmic PAC precursors in the current expression systems. PAC activity was significantly increased due to DegP-mediated processing in the periplasm. The strategy of DegP coexpression could be further applied to estimate the amounts of various PAC precursors and pac translational efficiency for different pac expression systems could be compared.