Chaperone-Mediated Folding and Maturation of the Penicillin Acylase Precursor in the Cytoplasm of Escherichia coli

Abstract

Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The outcome of proPAC folding and PAC maturation could be affected by several factors, such as inducer type, proPAC formation rate, and chaperone availability. Misfolding of proPAC in the cytoplasm could be partially resolved through the coexpression of cytoplasmic chaperones, such as trigger factor, GroEL/ES, or DnaK/J-GrpE. The three chaperones tested showed different extents of the effect on proPAC solublization and PAC maturation, and trigger factor had the most prominent one. However, the chaperone-mediated solublization of proPAC did not guarantee its maturation, which is usually limited by the first autoproteolytic step. It was observed that arabinose could act as an effective inducer for the induction of LL pac expression regulated by the lac-derived promoter system of trc. In addition, PAC maturation could be highly facilitated by arabinose supplementation and coexpression of trigger factor, suggesting that the coordination of chaperone systems with proper culture conditions could dramatically impact recombinant protein production. This study suggests that folding/misfolding of proPAC could be a major step limiting the overproduction of PAC in E. coli and that the problem could be resolved through the search for appropriate chaperones for coexpression. It also demonstrates the analogy in the issues of proPAC misfolding as well as the expression bottleneck occurring in the cytoplasm (i.e., LL pac expression) and those occurring in the periplasm (i.e., wild-type pac expression).

An Essential Role for the DnaK Molecular Chaperone in Stabilizing Over-expressed Substrate Proteins of the Bacterial Twin-arginine Translocation Pathway

“…DnaK, GroEL) and proteases (e.g. ClpXP, FtsH) play an important role in the early stages of Tat targeting and translocation in bacteria 25,36,38,39,45 and plants [55][56][57][58] (for a review see Fisher and DeLisa 34 ). Accordingly, we tested a panel of E. coli mutant strains lacking well-characterized chaperones or proteases and found that the molecular chaperone DnaK exerted the most significant effect on the expression and transport of synthetic Tat substrates such as ssTorA-GFP-SsrA, ssTorA-GFP and ssTorA-MBP.…”

“…34 Several lines of evidence implicate a role for general molecular chaperones in bacterial Tat transport including: (1) the chaperone trigger factor (TF) binds extensively to the TorA and SufI signal peptides, although depletion or over-expression of TF had little effect on the kinetics and efficiency of the Tat export process; 35 (2) GroEL was found to affect Tat-specific hydrogenase-1 maturation and assembly, 36 and was found to associate with the Tat-specific amidase AmiA; 37 (3) DnaK binds specifically to the Tat leader peptide of DmsA; 25 (4) members of the chaperone cascade including TF, DnaK-DnaJ-GrpE and GroEL interact with the Tat-specific REMP DmsD 38 ; and finally, (5) over-expression of DnaKDnaJ-GrpE, GroEL-GroES and TF has been shown to enhance the transport of the Tat substrate preproPAC. 39 In addition to the direct involvement of cytoplasmic chaperones, a number of indirect observations suggest that factors other than TatABC are involved in Tat transport. For instance, two separate groups have reported that translocation of the Tat substrate SufI could not be observed in inside-out inner membrane vesicles (INVs) prepared from wt E. coli cells.…”

High‐Level Gene Expression for Recombinant Penicillin Acylase Production Using the araB Promoter System in Escherichia coli

“…Such processing is usually overwhelmed by the excess amount of transiently formed proPAC upon high-level pac expression. The accumulation of the precursor, proPAC, in both soluble and insoluble fractions was previously identified as a major bottleneck limiting pac overexpression regulated by P trc and P T7 using IPTG as an inducer (24,31,35). Negative cellular responses, such as growth arrest and cell lysis, were also observed upon high-level pac expression.…”

“…The increased PAC activity upon degP S210A coexpression was mediated by the chaperone activity retained in DegP S210A , possibly via more effective proPAC processing, though the solubility of proPAC was not improved. The use of chaperones to improve the solubility and processing of proPAC was previously reported (35). On the other hand, the improved cellular physiology upon wild-type degP coexpression was mediated by DegP protease activity, which is inactivated for the mutant of DegP S210A .…”

Effect of heat‐shock proteins for relieving physiological stress and enhancing the production of penicillin acylase in Escherichia coli

“…DegP protease activity was primarily responsible for the improvement by degradation of abnormal proteins in the periplasm when pac was overexpressed (Pan et al, 2003). On the other hand, increasing the efficiency of the limiting step (i.e., posttranslational processing of proPAC) was recently demonstrated to be achievable by several cytoplasmic chaperone activities (Xu et al, 2005). The search for appropriate periplasmic chaperones that directly interact with periplasmic proPAC and suppress the periplasmic stress would provide hints for pinpointing the possible factors limiting pac overexpression.…”