Mutations in the translation initiation region of the pac gene resulting in increased levels of activity of penicillin G acylase.
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.
Department of Molecular Biology and Genetics, GYTE, Gebze Institute of Technology, 41400 Kocaeli, Turkey. ozlem@gyte.edu.tr
Özlem
Ö
Akkaya
Saliha Işsever
SI
Oztürk
Albert
A
Bolhuis
Füsun
F
Gümüşel
D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D009154:Mutation; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000235:genetics*; D014176:Protein Biosynthesis; D021381:Protein Transport; D012270:Ribosomes / Q000378:metabolism
D003062:Codon; D010399:Penicillin Amidase