Mutations in the translation initiation region of the pac gene resulting in increased levels of activity of penicillin G acylase

Abstract

Penicillin G acylase (PA) is an important enzyme used in the industrial production of b-lactam antibiotics. In this study, the effects of mutations in the translation initiation region of the Escherichia coli pac gene, encoding periplasmic PA, were examined. Several mutations led to increased amounts of PA activity, including those that lengthened the spacer region between the ribosome binding site and the ATG start codon, and those with altered codons on positions +2 and +4 relative to the start codon. These results indicated that the wild-type sequence of the pac gene does not provide maximum expression levels and that the strategies applied in this study can be used to improve production of PA in E. coli. Unexpectedly, our study also suggested that translocation of PA was, in contrast to earlier reports, shown not to require the Twin-arginine translocation pathway for transport into the periplasm.

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

“…In the case of the pac gene, encoding penicillin G acylase of E. coli , B. megaterium and other organisms [7,8], many different procedures have been employed to this end. The efficiency of transcription of the pac gene can be genetically modulated by mutation or removal of the regulatory region, mutations in the transcription initiation region, or by replacement of the pac promoter [9-11]. The post-translational processes, which are crucial for obtaining soluble active PGA, have been investigated to identify optimum host/vector combinations that can efficiently produce the mature protein [3,6], with co-expression of helper proteins that assist the formation of correctly-folded active PGA [6,12] .…”