The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the araB promoter (P-araB, also known as P-BAD) in Escherichia coli (E. coli). The current P-araB expression system exhibited minimum leaking pac expression in the absence of arabinose as well as fast and high-level pac expression upon induction with arabinose in a wide concentration range. The production of PAC was limited by the accumulation of PAC precursors (i.e., proPAC in both soluble and insoluble forms) and various negative cellular responses, such as growth arrest and cell lysis. The culture performance could be improved by degP coexpression and the individual contribution of DegP protease and chaperone activities to the enhancement on the production of PAC was characterized. The study highlights the importance of identifying the step(s) limiting high-level gene expression and subsequent design and construction of the host/vector system for enhancing recombinant protein production in E. coli.