We demonstrated the improvement of penicillin acylase (PAC) production by optimization of the host/vector system using genetic engineering strategies. Several expression plasmids with improved efficiency for the transcription of the pac gene and/or translation of the pac mRNA were constructed. Mutant strains, isolated by a novel screening method, were effective for use as the expression host to produce PAC. The feasibility of using the mutant strains harboring a selection of expression plasmids for the production of PAC was evaluated. The effect of the mutation(s) resulting in the improved PAC producing ability was characterized. While the production of PAC was significantly enhanced using the optimized host/vector system, the formation of PAC inclusion bodies was shown to be another seep limiting the production of recombinant PAC. (C) 2000 Elsevier Science Inc. All rights reserved.