Improving production of penicillin acylase in Escherichia coli via efficient DegP-mediated processing of precursors in periplasm

Citation
Yh. Lin et al., Improving production of penicillin acylase in Escherichia coli via efficient DegP-mediated processing of precursors in periplasm, PROCESS BIO, 37(1), 2001, pp. 23-30
Citations number
26
Categorie Soggetti
Biotecnology & Applied Microbiology","Biochemistry & Biophysics
Journal title
PROCESS BIOCHEMISTRY
ISSN journal
13595113 → ACNP
Volume
37
Issue
1
Year of publication
2001
Pages
23 - 30
Database
ISI
SICI code
1359-5113(200109)37:1<23:IPOPAI>2.0.ZU;2-1
Abstract
Penicillin acylase (PAC) from Escherichia coli has a complex enzyme formati on mechanism that is unusual for prokaryotic proteins. In this study, PAC i s used as a model protein to demonstrate an important concept of developing genetic strategies for improving recombinant protein production in E. coli ; namely, the bottleneck gene expression step(s) limiting recombinant prote in production must be precisely identified. Using the strong promoter syste m of trc for regulation of pac gene expression, the overproduction of PAC w as often limited by translocation and/or periplasmic processing steps, resu lting in intracellular accumulation of various types of PAC precursors. The over-accumulated PAC precursors formed inclusion bodies in the cytoplasm a nd/or periplasm. The periplasmic protease DegP could efficiently assist per iplasmic processing of PAC precursors and, therefore, the amount of peripla smic inclusion bodies was significantly reduced. The PAC inclusion bodies r emaining in DegP-coexpressing cells were located in the cytoplasm where the DegP function could not reach. These results indicate that the activity of DegP expression based on pKS12 was high enough for processing the over-acc umulated periplasmic PAC precursors in the current expression systems. PAC activity was significantly increased due to DegP-mediated processing in the periplasm. The strategy of DegP coexpression could be further applied to e stimate the amounts of various PAC precursors and pac translational efficie ncy for different pac expression systems could be compared. (C) 2001 Elsevi er Science Ltd. All rights reserved.