Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant Escherichiacoli Strains

Using periplasmic penicillin amidase (PA) from Escherichia coliATCC 11105 as a model recombinant protein, we reviewed the posttranslationalbottlenecks in its overexpression and undertook attempts toenhance its production in different recombinant E. coli expressionhosts. Intracellular proteolytic degradation of the newly synthesizedPA precursor and translocation through the plasma membrane weredetermined to be the main posttranslational processes limitingenzyme production. Rate constants for both intracellular proteolyticbreakdown (k_d) and transport (k_t) were used as quantitativetools for selection of the appropriate host system and cultivationmedium. The production of mature active PA was increased upto 10-fold when the protease-deficient strain E. coli BL21(DE3)was cultivated in medium without a proteinaceous substrate,as confirmed by a decrease in the sum of the constants k_d andk_t. The original signal sequence of pre-pro-PA was exchangedwith the OmpT signal peptide sequence in order to increase translocationefficiency; the effects of this change varied in the differentE. coli host strains. Furthermore, we established that simultaneouscoexpression of the OmpT pac gene with some proteins of theSec export machinery of the cell resulted in up to threefold-enhancedPA production. In parallel, we made efforts to increase PA fluxvia coexpression with the kil gene (killing protein). The primaryeffects of the kil gene were the release of PA into the extracellularmedium and an approximately threefold increase in the totalamount of PA produced per liter of bacterial culture.

INTRODUCTION

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Introduction

Escherichia coli is the most frequently used prokaryotic expressionsystem for high-level expression of homologous and heterologousproteins. However, despite its advantages, it has been commonlyobserved that the overexpression of foreign proteins in E. colitriggers a large metabolic burden and leads to undesired metabolicresponses, including changes in the central metabolism and regulatoryfunctions (36), cell growth retardation (1), and ribosome destructionand cell death (8). The expression of heterologous genes inrecombinant E. coli is associated with increased activity ofthe energy-generating dissimilatory pathway (41), and the fateof foreign proteins expressed in E. coli is determined in partby the degradative activities of the host cells (15). Moreover,the efficient expression of different genes in E. coli is nota routine matter, as the regulatory network of protein expressionat the posttranslational level is very complex and must be consideredin order to attain a high level of protein expression. Recentdevelopments in DNA recombinant technology have contributedto significant progress in protein overexpression. Genetic manipulationsimproving biosynthesis at the transcriptional level or optimizationof fermentation strategies developed to enhance the yield ofrecombinant proteins of interest lead to considerable increasesin their production. Nevertheless, higher levels of productionare still desired to satisfy the requirements for economicallyattractive process.

Using an industrially important enzyme, penicillin amidase (PA),from E. coli ATCC 11105 as a model system, we investigated theposttranslational yield-limiting steps influencing its productionand developed a biochemical engineering approach for enhancingits overexpression in recombinant E. coli strains. The nascentpolypeptide precursor is synthesized as 96-kDa pre-pro-PA (ppPA),containing, at its N terminus, a signal peptide which mediatestranslocation into the periplasm via the Tat pathway (18). Thereafter,translocated pro-PA (pPA) is further processed into two chains(A, 23 kDa, and B, 63 kDa) by various intra- and intermolecularautoproteolytic reactions (16, 21). Due to this complex regulationmechanism for biosynthesis, PA serves as a good model systemfor studying the relative influences of all of these processesin order to develop efficient strategies for improving recombinantprokaryotic protein production.

The emerging details of the structure and regulation of thepac gene (6, 32, 40) have allowed the manipulation of transcriptionaland translational efficiencies via the development of appropriatehost-vector systems (5). While PA activity was significantlyimproved, the processes leading to decreased yields posttranslationallywere not completely understood. Yields of active PA are restrictedby the following posttranslational processes: (i) nonspecificintracellular proteolytic degradation by host-specific cytoplasmicendopeptidases, (ii) inclusion body formation, (iii) transportthrough the cytoplasmic membrane, (iv) maturation, and (v) nonspecificproteolysis in the periplasm (17). The extent to which yieldsare affected by the posttranslational bottleneck steps mentionedabove suggests strategies for improvement of the overexpressionof a target protein.

In the present report, both translocation and intracellularproteolysis were manipulated in order to increase PA expressionat the molecular level in recombinant E. coli strains. Intracellularproteolysis was modulated either by the composition of the mediumor by the host strain used for overexpression. The exchangeof the original signal sequence of ppPA with the Sec-dependentOmpT signal peptide sequence varied in the different host strains.The overproduction of SecA, SecB, and SecF improved transportefficiency and enhanced up to threefold the level of expressionof an OmpT-PA fusion in the periplasm. Parallel efforts weremade to increase PA flux via coexpression with the kil gene(killing protein). The controlled release of PA into the extracellularmedium, leading to increased PA production, was only temporarydue to the decreased viability of the host cells.

MATERIALS AND METHODS

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Materials and Methods

DNA techniques.
The pac gene was amplified with its ribosome binding site (RBS)and Shine-Dalgarno sequence upstream of the start codon by usingchromosomal DNA from E. coli ATCC 11105 (Deutsche Sammlung vonMicroorganismen und Zellkulturen [DSMZ], Braunschweig, Germany)as a template, double digested with EcoRI and KpnI, and clonedinto the polylinker of the low-copy-number vector pMMB207 (Cm^r)(kindly provided by V. de Lorenzo); this procedure yielded plasmidpPAEC. Plasmid pPAOT, used for the in vivo expression of OmpT-PA,was constructed as described by Ignatova et al. (18). The pacgene in pPAEC or the OmpT pac gene in pPAOT was cloned underthe control of the tac promoter; therefore, the expression ofthese genes was induced with isopropyl-ß-D-thiogalactopyranoside(IPTG) (0.5 mM). For all genetic manipulations, plasmids weretransformed in E. coli DH5 ${alpha}$ cells. Transformations were carriedout according to the standard transformation procedure describedelsewhere (30) or by using an electroporator (PeqLab, Erlangen,Germany). pac-containing clones were selected on chloramphenicol(25 µg/ml)-containing Luria-Bertani (LB) agar plates andscreened phenotypically for PA activity (48).

The secB, secA, and secF genes were amplified from E. coli K5chromosomal DNA isolated by standard procedures as describedpreviously (30). The KpnI-BamHI-digested secB and secF geneswere inserted into the polylinker of pPAOT immediately afterthe stop codon of the OmpT pac gene, resulting in plasmids pPAOTsecBand pPAOTsecF, respectively. The XbaI-BamHI-digested PCR fragmentof secA was also subcloned into pPAOT, yielding plasmid pPAOTsecA.

The kil gene from the ColE1 plasmid (DSMZ) was amplified byusing 3-s dwell times with AGSGold polymerase on a Hybaid PCR-Sprintapparatus with active tube control according to the manufacturer'sinstructions (AGS-Hybaid GmbH, Heidelberg, Germany). The KpnI-BamHI-digestedkil amplification product was cloned into pPAEC after the stopcodon of the pac gene, resulting in plasmid pPAK2.

Bacterial strains and growth conditions.
The bacterial strains used in this study are listed in Table1. Transformed E. coli host strains were grown in LB medium,in M9 minimal medium (35) containing 2.5 g of glucose/liter(initial concentration), or in modified minimal medium (M9Ymedium) supplemented with 2.5 g of yeast extract/liter and 1g of glucose/liter. The selection marker for the plasmids testedwas chloramphenicol at 25 µg/ml. All experiments wereperformed at 28°C because of the temperature dependenceof intracellular proteolysis and its limitation on PA expression.

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TABLE 1. E. coli strains used in this study

Pulse-chase experiments.
Overnight E. coli K5 cultures were diluted 1:25 into M9 (asdescribed above) chase medium with a methionine-free amino acidmixture (50 µg/ml each) (pH 7.4) and cultivated at 28°C.As a selection marker, chloramphenicol was added at 25 µg/ml.At an A₆₀₀ of 0.4, the expression of ppPA and OmpT-PA was inducedwith 0.5 mM IPTG. At 10 min after induction, cells were pulse-labeledwith [³⁵S]methionine (100 µCi/ml; Amersham Buchler) for1 min at 28°C, followed by the addition of excess unlabeledmethionine (500 µg/ml) and chloramphenicol (100 µg/ml)(46). Mature PA was quantitatively isolated by immunoaffinitychromatography as described previously (18).

Cell fractionation.
After the cells were harvested by centrifugation at 1.93 x gfor 20 min, they were disrupted by sonication in phosphate buffer(pH 7.5; I = 0.01) (Branson sonifier W 450 for 10 min with a50% duty cycle at 4°C) or by a cold mild osmotic shock procedure(17). The periplasmic fraction was separated from the spheroplastsuspension by centrifugation at 8,000 rpm for 40 min at 4°C.The spheroplasts were sonicated in distilled water, and celldebris was removed by centrifugation at 13,000 rpm for 10 min.The clarified supernatant was designated the cytoplasmic fraction.

Measurement of in vivo proteolysis.
To study the rate of intracellular proteolysis in vivo, E. colihost strains transformed with plasmid pPAEC were grown underthe same conditions as those described above. At 3 h after inductionwith 0.5 mM IPTG, rifampin was added to the culture to a finalconcentration of 500 µg/ml to inhibit transcription initiationand to stop protein synthesis (37). Five-milliliter samplesof this culture were removed at different intervals and sonicated.Each sample was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis (PAGE) and Western blotting after centrifugationand cell pellet sonication.

Electrophoresis and immunoblotting.
SDS-PAGE of proteins with 10 or 12% polyacrylamide gels wasdone according to the method of Laemmli (24). Protein bandswere detected by Coomassie blue staining and immunoblotting(Immun-blot assay kit; Bio-Rad). Proteins were transferred topolyvinylidene difluoride membranes and detected with a monoclonalantibody for an epitope of the B chain of PA. The secondaryantibody used was goat anti-mouse immunoglobulin G (heavy andlight chains) conjugated to alkaline phosphatase (Bio-Rad).Detection was accomplished with 5-bromo-4-chloro-3-indolyl phosphateand nitroblue tetrazolium. PA with an isoelectric point of 7.0(0.1 mg/ml) and a pure mutant (Thr263Gly) pPA precursor (0.06mg/ml) were purified as described previously (20, 21) and usedas standards. Calculation of the relative optical densitiesof the bands of interest from the immunoblots, in relation tothe intensity of the standards, was performed by using the programONE-Dscan (Scananalytics, a division of CSPI, Billerica, Mass.).

Determination of enzyme activity.
PA activity in the homogenate or purified periplasmic fractionwas determined by a spectrophotometric assay with the chromogenicsubstrate 6-nitro-3-phenylacetamido benzoic acid. The changein the A₃₈₀ per minute was converted into benzylpenicillin units(BPU). Under these conditions for pure PA (1 mg/ml), the changein the A₃₈₀ was 3.0 per min, a value which corresponded to 42BPU/ml (20).

RESULTS

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Results

Influence of the medium on pac expression.
Cultivation of parental E. coli ATCC 11105 expressing the chromosomalpac gene in M9 minimal medium led to a 1.7-fold increase inPA yields compared to cultivation in complex LB medium (Table2). The cells grew slowly in M9 minimal medium, and the celldensity achieved was about twofold lower than that in LB medium(data not shown). Immunoblot analysis of E. coli ATCC 11105cells expressing PA showed an increased intensity of the B-chainband corresponding to active PA in the periplasmic fractionin M9 minimal medium compared to LB medium (Fig. 1). In thecytoplasmic fraction, some fragments (in the range of 60 to30 kDa) still possessing the epitope for the monoclonal antibodyused as the first antibody in the Western blots were detected.N-terminal amino acid analysis confirmed their origin as ppPA(data not shown). The 50-kDa proteolytic band dominated andwas used in further experiments as a representative exampleto monitor the changes in the degradation activity of host cellsunder the studied conditions. N-terminal amino acid analysisof the 50-kDa band revealed the sequence AEKPGYYL, which overlapsamino acids 347 to 354 in the B chain (numbering is accordingto the published primary amino acid sequence of pPA) (42) andcorresponds to the calculated molecular mass of 53.6 kDa forthe fragment Ala₃₄₇-Arg₈₂₀. A significantly decreased intensityof the 50-kDa band was observed in M9 minimal medium (Fig. 1B)compared to the intensity in the cytoplasmic fraction of cellscultivated in complex LB medium (Fig. 1A). The intensity ratioof mature PA to proteolytic products increased for M9 minimalmedium compared to LB medium when larger parts of the newlysynthesized precursor underwent intracellular proteolytic degradation,leading to increased intensity of the main proteolytic band(50 kDa).

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TABLE 2. PA expression levels in various E. coli host cells^a

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FIG. 1. Influence of the medium on PA biosynthesis in parental strain E. coli ATCC 11105. Cells were cultivated in LB medium (A) or M9 minimal medium (B) at 28°C. Aliquots were removed at the indicated times after the addition of phenylacetic acid (1 g/liter), separated into cytoplasmic (C) and periplasmic (P) fractions, resolved by SDS-PAGE (12.5% gel), and immunoblotted. For each time point, the same number of cells was analyzed. Pure PA at a concentration of 0.1 mg/ml (B chain) and the slowly processed Thr263Gly mutant PA precursor (pPA) at a concentration of 0.06 mg/ml were used as standards.

Screening for an appropriate expression system.
For selection of an appropriate host expression system, differentE. coli strains (Table 1) were cultivated in complex and minimalmedia (Table 2). Previous experiments showed that PA productioncorrelates with cell growth and that PA biosynthesis stops inthe early stationary phase (17). pac expression was inducedin the middle of the logarithmic phase; at approximately 6 hafter induction, the cells reached the stationary phase. Themeasured specific activity at this point was used for quantitativecomparisons (Table 2). The levels of expression of PA in allstrains tested (except for DH5 ${alpha}$ ) harboring plasmid pPAEC in LBmedium differed up to twofold from that in E. coli ATCC 11105.In the proteinase-deficient host strain E. coli BL21(DE3), themeasured PA activity was comparable to that in control strainE. coli ATCC 11105 cultivated in M9 minimal medium (Table 2).A substantial increase in PA activity was measured when E. coliBL21(DE3) cells were cultivated in medium without proteinaceoussubstrates, i.e., M9 minimal medium (Table 2). This expressionlevel was approximately 10-fold higher than that in controlstrain E. coli ATCC 11105 cultivated in the same medium. E.coli BL21(DE3) cells grew more slowly in M9 minimal medium thanthe other host strains and had an extended lag phase in M9 minimalmedium. The achievement of the stationary phase in shake flasksoccurred approximately 10 h after induction, necessitating longercultivation of these host cells.

In order to quantify the influence of posttranslational processeson protein flux, rate constants for intracellular proteolysis(k_d) and for transport through the membrane (k_t) must be determined.The kinetic equation for the in vivo fate of an intracellularprotein precursor (P) can be expressed as follows:

(1)
where k_s is the rate constant for the synthesisof mRNA, k_d is the rate constant for intracellular proteolyticdegradation, and [P]_c is the protein concentration in the cytoplasmicat any time. In all degradation processes, proteolytic enzymesfrom the destructive machinery of the cell are involved. Therefore,the apparent first-order rate constant k_d is a summarizing constantthat includes the Michaelis-Menten constant K_m, the turnovernumber k_cat, and the concentration of peptidases participatingin this process, assuming that the intracellular peptidasesare not saturated.

For proteins which are not localized in the cytoplasmic spaceand which are additionally translocated through the membrane,such as PA, the equation is changed as follows:

(2)
where k_t is the apparent first-order rate constantfor transport through the plasma membrane and [ppPA]_c is theprecursor concentration in the cytoplasm at any time. Assumingthat the translocation machinery is not saturated, i.e., a first-orderprocess, k_t depends on the concentration of ppPA and on thebinding and efficiency of the translocation system.

The sum of the apparent first-order rate constants for proteolyticdegradation and transport (k_d plus k_t) can be determined afterthe inhibition of de novo protein synthesis. Equation 1 is thustransformed as follows:

(3)

For this experiment, de novo protein synthesis was blocked withrifampin; at regular intervals, samples were withdrawn and subjectedto immunoblot analysis (Fig. 2). Protein synthesis flux wasnot abolished immediately due to a delay in the diffusion ofthe antibiotic into the cells, leading to a slight increasein the intensity of the ppPA band in the first 20 min. Thereafter,the intensity of the ppPA band decreased continuously parallelto the observed concomitant increase in the intensity of themain proteolytic band (50 kDa) (Fig. 2). The degradation productsobtained from the intracellular breakdown of the ppPA precursorresulted from both primary and secondary cleavages involvingintracellular peptidases. Proteolytic fragments still containingthe N-terminal signal peptide of ppPA might have competed forthe translocation pores in the membrane and influenced the estimationof k_t. Thus, the sum of k_t and k_d is the only quantitative measurefor comparing the strains used in this study. The similar PAactivities measured for host strains E. coli K5, E. coli MC4100,and E. coli BL21(DE3) cultivated in LB medium (Table 2) correlatedwith the calculated similar values for the sum of k_d and k_tfor that medium (Table 3). In this case, the intensity of themain protein degradation band (50 kDa) was lower in E. coliBL21(DE3) cells (Fig. 2A) than in E. coli K5 cells (Fig. 2C)or in E. coli MC4100 cells (Fig. 2B), indicating reduced intracellularproteolysis in E. coli BL21(DE3) cells.

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FIG. 2. In vivo proteolysis of ppPA in E. coli host cells. E. coli BL21(DE3) (A), E. coli MC4100 (B), and E. coli K5 (C) were used as host cells. Residual concentrations of ppPA and the 50-kDa proteolytic product were determined by immunoblot analysis after the inhibition of de novo protein synthesis by rifampin. The relative optical densities (dens.) of the bands of interest from the Western blot were quantified by using ONE-Dscan. ppPA density was related to the density of the pPA standard, and the density of the 50-kDa proteolytic band was related to the density of the PA standard (for details, see Materials and Methods). LB, LB medium; M9, M9 minimal medium.

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TABLE 3. Rate constants for intracellular proteolysis and transport in different strains^a

The omission of the proteinaceous substrates by changing themedium to M9 minimal medium led to a significant reduction inthe amount of the main proteolytic band (50 kDa) in E. coliBL21(DE3) harboring pPAEC (Fig. 2A) and caused a decrease inthe sum of k_d and k_t to 0.28 h^-1 (Table 3). The effect of thedecreased sum of k_d and k_t can quantitatively explain the observedincrease in PA activity in E. coli BL21(DE3) harboring (pPAEC)compared to wild-type E. coli ATCC 11105 (Tables 2 and 3).

Improvement of the translocation capacity of the host cell.
Recently published investigations indicated that the parentalsignal peptide of E. coli PA directs ppPA to the Tat translocationpathway (18). Compared to the velocity of Sec-dependent exportof proteins, the translocation of proteins by Tat-mediated transportis slower (2, 7). In this context, in order to accelerate exportand to decrease the exposure of the ppPA precursor in the cytoplasm,we exchanged the original signal peptide of ppPA with a Sec-dependentsignal peptide (from the OmpT peptide); this procedure efficientlyrerouted the OmpT-PA fusion to the Sec pathway (18). In E. coliK5, the kinetics of translocation of the OmpT-PA precursor werelinear over the first 2 min, and translocation was accomplishedafter 5 min (Fig. 3B and C) The translocation of OmpT-PA wasabout threefold faster than the transport of ppPA (Fig. 3A).Similar translocation kinetics for both precursors were alsoobserved for host strain E. coli MC4100 (18).

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FIG. 3. Translocation kinetics for ppPA and OmpT-PA. (A and B) E. coli K5 carrying plasmid pPAEC or pPAOT was grown at 28°C in the chase medium. At 10 min after induction, the cells were pulse-labeled with [³⁵S]methionine for 1 min and then chased with nonradioactive methionine. At the times indicated, samples (500 µl) were withdrawn, subjected to immunoaffinity chromatography for the quantitative isolation of mature PA, and assessed by SDS-PAGE followed by autoradiography. The position of the B-chain PA band was verified by using a prestained protein marker (Bench Mark; Gibco BRL). (A) ppPA. (B) OmpT-PA. (C) The intensity of the B chain of PA from panel A (squares) and panel B (circles) was quantified by using ONE-Dscan and is represented graphically as a function of the chase time. The highest intensity reached in each blot was taken as 1. Error bars indicate standard deviations.

As has been already shown for the isolated main degradationproduct (50-kDa band), the intracellular proteolytic attackon ppPA occurs at residues localized in the B chain. ppPA andOmpT-PA differ only in their signal sequences, allowing us toassume that the same intracellular proteolytic degradation occursfor PA with different signal peptides. To examine the influenceof the signal peptide on intracellular proteolysis, we transformedplasmid pPAOT into different E. coli host strains (Table 4).A comparison of the final PA activities achieved by expressingplasmid pPAOT (Table 4) with the pPAEC vector (Table 2) revealedincreased enzyme activities only in E. coli DH5 ${alpha}$ , E. coli K5,and E. coli MC4100. In contrast, the levels of expression ofOmpT-PA in E. coli JM109 decreased by 50% in LB medium and by11% in M9Y medium. The situation was less clear for E. coliBL21(DE3), for which we detected approximately a twofold reductionin PA activity in all media tested (Table 4).

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TABLE 4. OmpT-PA expression levels in various E. coli host cells^a

Another approach to manipulating translocation is to increasethe export efficiency of the cell. To determine whether theoverexpression of Sec proteins might enhance the PA expressionlevel, E. coli K5 cells were transformed with a plasmid bearingadditional copies of either secA, secB, or secF cloned behindthe OmpT pac gene under the control of the same tac promoter.The overproduction of SecA had no significant influence on PAproduction. Although an increase in PA activity in the periplasmicfraction was measured (Fig. 4A), the intensity of the PA bandin the immunoblot was comparable to that in the control experiment(Fig. 4B). The coexpression of OmpT-PA with SecB and SecF ledto three- and twofold-enhanced PA expression, respectively (Fig.4).

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FIG. 4. Coexpression of OmpT-PA with some of the compounds of the Sec machinery. (A) E. coli K5 host cells transformed with the indicated plasmids were grown in LB medium supplemented with chloramphenicol (25 µg/ml) at 28°C to an A₆₀₀ of 0.6. IPTG (0.5 mM) was added, and incubation was continued for 6 h. The activity of PA in the control E. coli K5 culture transformed with pPAOT was taken as 1. Error bars indicate standard deviations. (B) Western blot of the periplasmic fraction of the same samples as those used for activity measurements in panel A. The protein concentration load per lane corresponds to the same amount of cells verified by optical density measurements. Pure PA (0.1 mg/ml) was used as a standard (PA_st).

Excretion of PA into the extracellular medium.
Several homologous and heterologous proteins of different originsare efficiently released into the culture medium by the actionof the kil gene of the ColE1 plasmid. The small protein productof that gene (6 kDa) resembles the E. coli membrane lipoproteincausing the release of periplasmic proteins (44). We assumedthat by guiding protein flux into the extracellular medium,the cellular machineries (e.g., translocation pore and periplasmwith periplasmic inclusion bodies) saturated by the overproducedprotein could be desaturated. Therefore, there were reasonsto believe that the controlled release of PA into the mediumcould also enhance its overproduction. To examine this notion,we placed both the target pac gene and the kil gene in the sameplasmid, pPAK2, under the control of the tac promoter. Culturesof E. coli K5(pPAK2) and E. coli K5(pPAEC) as a kil-negativecontrol were grown at 28°C in LB medium. Over the first3 h after induction, the increase in PA activity in the cellsand this released into the periplasm was linear and almost threefoldhigher than that in the control (Fig. 5A). Over the next 3 h,PA activity measured in the extracellular medium continued toincrease, whereas cellular PA activity dropped and the totalamount of PA produced per liter of bacterial culture was 25%higher than that measured in the control. At that time, kil-bearingcells stopped de novo ppPA synthesis and released the alreadysynthesized mature PA from the periplasm into the culture broth.During the last 3 h, cell growth was inhibited and the numberof viable cells decreased slightly, as determined by countingof colonies on selective agar plates (data not shown).

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FIG. 5. Release of PA into the extracellular medium. (A) E. coli K5 cells transformed with pPAEC (control) and pPAK2 were grown at 28°C in LB medium supplemented with 25 µg of chloramphenicol/ml. Plasmid-carried pac and kil genes were induced with 0.5 mM IPTG; thereafter, at the times indicated, 1-ml samples were withdrawn. The PA activity in kil-expressing cells was measured in homogenized cells (periplasm) and in the culture supernatant (extracellular). (B) E. coli K5(pPAK2) cells were cultivated in different media as described for the cultivation in panel A. At 6 h after IPTG induction, PA activity was measured in the supernatant (extracellular) and in homogenized cells (periplasm). LB, LB medium; M9, M9 minimal medium.

To test whether the beneficial effect of the media on PA biosynthesis(Table 2) might have a similar influence on cells bearing thekil gene, E. coli K5(pPAK2) cells were also cultured in M9 minimalmedium and M9Y medium. The change in the cultivation mediumled to a moderate release of extracellular PA (Fig. 5B) and,unexpectedly, the detected total PA activity was lower thanthe activity measured for E. coli K5(pPAEC) (Table 2). Despitethat result, the viability of the kil-induced cells was notreduced.

DISCUSSION

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Discussion

A primary goal of industrial technology for the overexpressionof proteins in recombinant E. coli hosts is the cost-effectiveincrease in the production of the desired protein. This canbe achieved by increasing the production of the active proteinper cell and/or by manipulation of the fermentation conditions.Optimization of the fermentation media and operational strategiesor a combination of both succeeded in enhancing the productionof some recombinant proteins (15, 19, 47). Increasing proteinproduction by means of increasing cell density for recombinantE. coli host strains has reached its limits (47).

Alternatively, genetic engineering methods influencing the levelsof expression of genes offer more possibilities for improvingthe biosynthesis of recombinant proteins. Some are specificfor each gene, such as mRNA stability, codon usage, and transcriptionalregulation in the 5'- and 3'-flanking regulatory regions. Theseparameters must be optimized individually. Another, more generalmethod for improving productivity is the use of strong and tightlyregulated promoters (e.g., tac and rhaBAD). However, this methodoften leads to an imbalance of protein flux, causing aggregationof the product in insoluble inclusion bodies. The problem withcytoplasmic inclusion bodies can be overcome by the applicationof low-copy-number plasmids for the overexpression of proteins.For periplasmic proteins, insoluble inclusion bodies composedof protein precursors are localized in the cytoplasm and periplasm(4, 25). The amounts of periplasmic inclusion bodies can besignificantly reduced by coexpression with periplasmic proteases(27).

Besides optimizing recombinant protein production at the translationalor transcriptional level, many posttranslational factors (e.g.,folding and maturation of the protein and physiological activitiesof the host) influence the level of biosynthesis. The expressionof foreign proteins in E. coli causes a metabolic burden oftenresulting in increased degradative activities of the host. Thebiotechnological consequences of intracellular host-dependentproteolysis are usually loss of the product and its contaminationby proteolytic products. A variety of strategies have been developedto reduce the intracellular proteolytic destruction of recombinantproteins, e.g., reduction of the growth rate of cells, protectivefusions, and replacement of specific residues to eliminate cleavagesites (for a review, see reference 13). For the system usedin this study, PA, intracellular proteolysis causes more thanan 80% loss of a newly synthesized ppPA precursor for parentalE. coli ATCC 11105 (17). Thus, the use of a proteinase-deficientstrain as a host strain for pac expression seemed to be a successfulstrategy for improvement of the yield of proteolytically sensitiveprecursors. For this purpose, we chose commercially availableE. coli BL21(DE3), which has natural deficiencies of ATP-dependentproteinase Lon and outer membrane proteinase OmpT (14). Lonand two other proteases (ClpAP and ClpXP) are responsible for70 to 80% of the energy-dependent protein degradation in E.coli (31). Nevertheless, attempts to increase sufficiently PAproduction in BL21(DE3) as a host strain cultivated in complexLB medium failed. The protease activity of the host is influencedby the medium composition, and cultivation of E. coli BL21(DE3)harboring pPAEC in medium without proteinaceous substrates increasedPA production by up to 10-fold. Moreover, the amount of themain proteolytic band (50 kDa) was reduced, and the calculatedrate constants for transport and intracellular proteolysis (k_dand k_t) decreased to 0.28 h^-1. The intracellular proteolyticbreakdown could not be circumvented completely but was reduceddrastically. Similar results have been observed in expressionstudies with Bacillus subtilis, showing that even with strainsmissing extracellular proteases, proteolytic degradation reducesthe yield of the protein of interest (3). In some recently publishedstudies, it was observed that PA production is sensitive tocomplex proteinaceous substrates and that yields can be raisedwhen minimal medium with different carbon sources is used (29,32, 43).

For proteins destined to be exported into the periplasm, thetranslocation machinery of the host markedly restricts theiryields (45). The efficient transfer of preproteins through membranesrequires an intact translocation system. However, when a proteinis overexpressed, the export machinery becomes overloaded andits efficiency decreases significantly, and some advantagesof the system are lost. An approach for influencing the bottlenecksteps in secretion is the manipulation of the signal peptide,the sole determinant directing preproteins to the appropriatetranslocation system (45). Optimization of original signal peptideshas led to improved yields of many periplasmic enzymes (12,28). In experiments with the OmpT-PA fusion, the effect variedin the different host strains, and no consistent influence couldbe found. The exchange of the whole signal peptide is not anappropriate approach for enhancing extracellular protein production,opposite the use of single mutations in the original signalsequence of the protein of interest (12).

Alternatively, providing a strain with additional copies ofcomponents of the translocation system can relieve secretionand enhance the yields of heterologous proteins (38). The overexpressionof SecYEG proportionally enhances in vitro Sec translocase activity(9, 10). Perez-Perez et al. (38) reported that supplementationwith a plasmid bearing copies of secE and prlA4 (secY allele)increased the periplasmic production of a preOmpA-interleukin-6fusion about 10 times. The coexpression of an OmpT-PA fusionwith some of the components of the Sec machinery (e.g., SecA,SecB, and SecF) led to an overall increase in PA activity inthe periplasmic fraction. Both SecA and SecB were chosen basedon the assumption that as components of the Sec machinery deliveringpreproteins to the translocation pore in the membrane (11),they might prevent the cytoplasmic proteolytic breakdown ofppPA. SecD and SecF are important in the release of the proteininto the periplasm (39). The most remarkable influence was observedfor coexpression with SecB. The SecB protein, which is involvedin the early translocation act, maintains presecretory proteinsin a translocation-competent state (23). Obviously, for PA,the chaperone function of SecB stabilizes ppPA against intracellularproteolytic degradation, and more precursor is delivered tothe translocation pore.

Recently published results demonstrated that controlled fluxof a target protein into the extracellular medium by coexpressionwith the kil gene can increase its total production (34). However,the use of strong inducible promoters causes some problems withthe viability of recipient cells. For the model system usedin this study, the effect of kil gene expression not only variedin the different media but also depended, as recently reported,on the host strain used for pac expression (26). As a consequence,even though coexpression of the protein of interest with thekilling protein could enhance protein yield, the observed drawbacksdemanded optimization of the system by use of different conditionsto attain the desired goals. The use of simultaneous inductionof the kil gene with the gene for the recombinant protein isnot an efficient strategy, as the viability of cells after inductionis severely reduced. Other promoters, e.g., weak constitutiveor growth-phase-dependent promoters (22, 33), enable viabilityto be maintained efficiently. However, the expression of thekil gene under the control of a stationary-phase-dependent promoterhas not given the expected increase in the yield of the targetprotein, as adaptation to the stationary phase requires thereduction of global gene expression (33). The use of separateand differently controlled promoters, a strong one for the recombinantprotein and a moderate one for the killing protein, might preventearly lysis of host cells without the loss of their expressioncapabilities.

As outlined above, several posttranslational bottlenecks mustbe considered for achieving improvement of the yields of recombinantproteins in E. coli. Thus, the efficient production of a targetprotein requires the identification and quantification of specificyield-limiting processes followed by a direct attempt to decreasetheir influence. Quantification of the rates of proteolysisand transport through the membrane for the model system usedhere, PA, allowed us to conclude that the use of proteinase-deficientstrains as host strains cultivated in medium without proteinaceoussubstrates is a successful strategy for achieving higher productivityof a proteolysis-susceptible target protein. Furthermore, influencingtranslocation offers ample possibilities for improving proteinyield. Thus, the development of better strategies designed tomaximize the yields of recombinant proteins must be based onsimultaneous modulation of more than one of the yield-restrictingfactors.

ACKNOWLEDGMENTS

We thank St. Stoeva (Abteilung Physiologie und Biochemie, UniversitätTübingen) for performing the N-terminal sequence analysisand P. Zipfel (Jena, Germany) for making it possible for usto perform the pulse-chase experiments in his laboratory.

This work was funded by DFG (KA 505/9-1).

FOOTNOTES

* Corresponding author. Mailing address: Institut für Biotecnologie II, Technische Universität Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany. Phone: 49 40 42878 2204. Fax: 49 40 42878 2127. E-mail: ignatova{at}tuhh.de.

REFERENCES

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