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
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.