Genetic manipulation to identify limiting steps and develop strategies forhigh-level expression of penicillin acylase in Escherichia coli

We have identified the bottleneck steps limiting expression of penicillin a cylase (PAC) through comparison of the expression performance for various P AC-expression vectors constructed by genetically modulating the efficiencie s of transcription and/or translation of the pac gene. To our knowledge, th is is the first report demonstrating that expression of PAC could be limite d by various steps, such as transcription, translation, and post-translatio nal steps (i.e, translocation and periplasmic processing), depending on the host/vector systems. Results also indicate that the structure of the wild- type pac gene might not be optimal for direct use in production of PAC usin g recombinant DNA technology. To improve the gene expression, transcription was enhanced by manipulating certain DNA bases in the pac regulatory regio n, whereas translation was enhanced by enlarging the spacing between the ri bosome binding site and the ATG initiation codon to increase the initiation efficiency. The information is useful in terms of developing genetic strat egies for overproduction of recombinant PAC in Escherichia coli. (C) 1999 J ohn Wiley & Sons, Inc. Biotechnol Bioeng 63: 283-272, 1999.