Cytoplasmic overexpression, folding, and processing of penicillin acylase precursor in Escherichia coli

Penicillin acylase (PAC) precursor, proPAC, was overproduced in a soluble or insoluble form in the cytoplasm of Escherichia coli through the expression of the leader-less pac gene (ll-pac) devoid of the coding region for the signal peptide of PAC. Also, a portion of the overexpressed proPAC was further processed to form mature PAC, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The cultivation performance for ll-pac expression was limited by several factors, including (1) misfolding of proPAC, resulting in the aggregation of insoluble proPAC as inclusion bodies, (2) intracellular proteolysis, leading to the degradation of the overexpressed gene products, and (3) inefficient PAC maturation, limiting the formation of active PAC. The effect of coexpression of various cytoplasmic chaperones, including trigger factor, GroEL/ES, DnaK/J-GrpE, and their combinations, on ll-pac expression was investigated. Intracellular proteolysis of the overexpressed gene products could be prevented by coexpression of GroEL/ES. On the other hand, coexpression of trigger factor appeared to be able to facilitate the folding of soluble proPAC and to improve PAC maturation. The roles of trigger factor and GroEL/ES could be coordinated to significantly improve ll-pac expression performance. DnaK/J-GrpE had an effect for solublization of proPAC and perhaps, similar to trigger factor, for improving PAC maturation. The ll-pac expression performance was also significantly improved through the simultaneous coexpression of DnaK/J-GrpE and GroEL/ES. The results of the study suggest that the folding and/or processing of proPAC could be a major issue limiting the overproduction of PAC in E. coli and the bottleneck could be eliminated through the coexpression of appropriate chaperone(s).     

DegP-coexpression minimizes inclusion-body formation upon overproduction of recombinant penicillin acylase in Escherichia coli

We demonstrated the enhancement of recombinant penicillin acylase (PAC) production in Escherichia coli by increasing the intracellular concentration of the periplasmic protease DegP. Using appropriate host/vector systems (e.g., HB101 harboring pTrcKnPAC2902 or MDDeltaP7 harboring pTrcKnPAC2902) in which the expression of the pac gene was regulated by the strong trc promoter, the overproduction of PAC was often limited by periplasmic processing and inclusion bodies composed of protein aggregates of PAC precursors were formed in the periplasm. The amount of these periplasmic inclusion bodies was significantly reduced and PAC activity was significantly increased upon coexpression of DegP. The specific PAC activity reached an extremely high level of 674 U/L/OD(600) for MDDeltaP7 harboring pTrcKnPAC2902 and pKS12 under optimum culture conditions. However, such improvement in the production of PAC was not observed for the expression systems (e.g., MDDeltaP7 harboring pCLL2902) in which the periplasmic processing was not the step limiting the production of PAC. The results suggest that DegP could in vivo assist the periplasmic processing though the enzyme is shown to be not absolutely required for the formation of active PAC in E. coli. In addition, the steps limiting the production of PAC are identified and the reasons for the formation of PAC inclusion bodies are discussed here.     

Role of the PDZ domains in Escherichia coli DegP protein

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.     

Permeabilization of Escherichia coli cells for enhanced penicillin acylase activity

Summary Escherichia coli cells with penicillin acylase activity were permeabilized with aqueous solutions of the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB), at pH 8.0 and the activity was found to have almost doubled. The concentration of CTAB, the time and temperature of treatment were optimised for maximum enzyme activity and were found to be 0.2%, 20 min and 5°C respectively. Subsequently, the cell bound activity was retained for a longer period by chemical cross-linking with 0.1% glutaraldehyde.     

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

粪产碱杆菌青霉素G酰化酶的分离提取

比较研究了几种破碎大肠杆菌细胞的方法,如渗透压法、超声波法、玻珠震碎法、玻珠研磨法、有机溶剂法、冻融法以及盐酸胍/EDTA法等,以确定出一种简单、快速、高效的破碎重组大肠杆菌细胞的方法获得粪产碱杆菌青霉素G酰化酶(AfPGA)用于后续试验。结果表明玻珠震碎法、超声波法和渗透压法是较优的细胞破碎方法,活力回收率分别为99.7%、78.4%、60.7%,其他方法均低于22%。而比活力以渗透压法为最高,达到4.40 U/mg。     

Recombinant protein folding and misfolding in Escherichia coli

The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding. Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form. The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E. coli will remain an important host for the production of both commodity and high value added proteins.     

Immobilization of permeabilized Escherichia coli cells with penicillin acylase activity.

Escherichia coli cells with penicillin acylase activity were sequentially treated at pH 7.8 with aqueous solutions of N-cetyl-N,N,N-trimethylammonium bromide and glutaraldehyde and then immobilized within porous polyacrylamide beads. The immobilized whole cells showed enhanced hydrolysis rates in the conversion of benzylpenicillin to 6-aminopenicillanic acid (6-APA) compared to untreated cells immobilized and used under identical conditions. The immobilized system showed no apparent loss in enzyme activity when used repeatedly over 90 cycles for 6-APA production from 4% benzylpenicillin.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Arabinose-induction of lac-derived promoter systems for penicillin acylase production in Escherichia coli

Arabinose was shown to serve as an effective inducer for induction of the lac-derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-beta-d-thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by the trc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac-derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LL pac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression.     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Characterization of the T7 promoter system for expressing penicillin acylase in Escherichia coli

The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the T7 promoter in Escherichia coli. PAC, with its complex formation mechanism, serves as a unique target protein for demonstration of several key strategies for enhancing recombinant protein production. The current T7 system for pac overexpression was fraught with various technical hurdles. Upon the induction with a conventional inducer of isopropyl-β-d-thiogalactopyranoside (IPTG), the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies and various negative cellular responses such as growth inhibition and cell lysis. The expression performance could be improved by the coexpression of degP encoding a periplasmic protein with protease and chaperone activities. In addition to IPTG, arabinose was shown to be another effective inducer. Interestingly, arabinose not only induced the current T7 promoter system for pac expression but also facilitated the posttranslational processing of proPAC for maturation, resulting in significant enhancement for the production of PAC. Glycerol appeared to have an effect similar to, but not as significant as, arabinose for enhancing the production of PAC. The study highlights the importance of developing suitable genetically engineered strains with culture conditions for enhancing recombinant protein production in E. coli.     

Effect of pH on high-temperature production of bacterial penicillin acylase in Escherichia coli

High-temperature-oriented production of bacterial penicillin acylase (PAC), which is usually expressed at low temperatures (less than 30 degrees C), was demonstrated in this study via heterologous expression of the Providencia rettgeri (P. rettgeri) pac gene in Escherichia coli (E. coli). While it is possible to produce PAC at a temperature as high as 37 degrees C, the environmental condition (specifically, culture pH) critically affected culture performance. Production of PAC at 37 degrees C was feasible only when culture pH was close to neutral (i.e., 6.5-7.5). Outside this pH range, cell physiology for the host/vector system was seriously affected, resulting in poor culture performance. In acidic culture environments, temperature significantly affected the pac expression level and specific PAC activity decreased with an increase in culture temperature. In basic culture environments, cell growth was seriously inhibited though the pac expression level was minimally affected by temperature. Such unusual types of pH and temperature effects on pac expression were never reported for bacterial PACs. The results suggest that culture pH should be precisely controlled for the current host/vector systems being applied on the overproduction of P. rettgeri PAC in E. coli at high temperatures.     

Effects of lipoprotein overproduction on the induction of DegP (HtrA) involved in quality control in the Escherichia coli periplasm

Recent biochemical examination has revealed the presence of at least 90 different lipoproteins in Escherichia coli. Among previously identified lipoproteins, only an outer membrane lipoprotein, NlpE, is known to induce expression of the degP gene upon its overproduction. The degP gene encodes a periplasmic protease, which is thought to be involved in the digestion of unfolded proteins, and is essential for growth at high temperatures. However, it is not completely clear why NlpE overproduction causes degP expression. Moreover, among newly confirmed lipoproteins, there may be others that also induce degP expression. Therefore, we overproduced each of the 90 lipoproteins and examined the level of degP expression as beta-galactosidase activity by using a degP promoter-lacZ fusion. The extent of degP expression caused by NlpE overproduction was dependent on the mode of degP-lacZ fusion. On the other hand, new inner membrane lipoprotein YafY strongly induced degP expression irrespective of the mode of fusion even though the level of overproduced YafY was lower than that of NlpE. The induction of degP expression by YafY overproduction was dependent on the Cpx two-component system. Alteration of the lipoprotein-sorting signals of NlpE and YafY did not abolish the degP induction. However, a YafY derivative possessing the outer membrane signal remained on inner membranes. The non-lipidated derivative of NlpE did not induce degP expression, indicating that membrane anchoring is essential for degP induction. The amino acid sequences of YafY and YfjS, another inner membrane lipoprotein, are highly identical, but overproduction of the latter did not induce degP expression. Construction of various YafY-YfjS chimeric lipoproteins revealed that only a few residues located in the N- and C-terminal regions were important for the induction of DegP.     

An improved method to clone penicillin acylase genes: Cloning and expression in Escherichia coli of penicillin G acylase from Kluyvera citrophila

A simple and versatile procedure to clone penicillin acylase genes has been developed. It involves the construction of a plasmid library in a host presenting an amino acid auxotrophy. Recombinant clones carrying the acylase gene were selected on a minimal medium containing instead of the required amino acid its phenylacetyl derivative. Penicillin acylase genes from Escherichia coli ATCC 11105 and Kluyvera citrophila ATCC 21285 have been cloned in E. coli using this technique. The restriction map of the region containing the E. coli penicillin acylase gene was found to be similar to that described by H. Mayer et al. (in: Plasmids of Medical, Environmental and Commercial Importance (Timmis, K.M. and Paler, A., eds.), pp. 459–470, Elsevier, Amsterdam 1979). K. citrophila acylase gene was located within a 3.0 kb Hind III-PvuI fragment. Some differences were observed between the partial restriction maps of both genes. In addition, the production of those clones carrying the E. coli acylase was more sensitive to the growth temperature than that of the clones containing the K. citrophila gene. Bacteria harbouring plasmids containing the K. citrophila acylase sequence were able to produce about 30 fold more enzyme than the parental strain. A 60 000 dalton polypeptide corresponding to the K. citrophila acylase has been detected in a maxicell system. The industrial applications of the procedure are discussed.     

Chaperone-mediated folding and maturation of the penicillin acylase precursor in the cytoplasm of Escherichia coli

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).