Strain improvement to enhance the production of recombinant penicillin acylase in high-cell-density Escherichia coli cultures

Using fed-batch operation for high-cell-density cultivation, efforts are frequently made for optimization of culture parameters, particularly feeding strategy. The current study also emphasized the importance of selecting strains for the production of recombinant proteins in high-cell-density cultures. With Escherichia coli penicillin acylase (PAC) as a target protein, the host/vector system of MDdeltaP7 harboring pTrcKnPAC2902 and pKS12 was designed for optimization of fed-batch cultivation for recombinant protein production. The host, MDdeltaP7, potentially had a high translational and periplasmic processing efficiency for pac expression. On the other hand, the vector, pTrcKnPAC2902, was genetically constructed for pac overexpression. Coexistence of the other vector, pKS12, significantly enhanced PAC production by improving cell physiology and reducing the amount of inclusion body formation upon pac overexpression. An extremely high volumetric PAC activity at 37,500 U/L was obtained with the use of the developed host/vector system under optimum fed-batch culture conditions.     

Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.     

Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 2. Redirection of metabolic fluxes

The impact of temperature-induced synthesis of human basic fibroblast growth factor (hFGF-2) in high-cell-density cultures of recombinant Escherichia coli was studied by estimating metabolic flux variations. Metabolic flux distributions in E. coli were calculated by means of a stoichiometric network and linear programming. After the temperature upshift, a substantially elevated energy demand for synthesis of hFGF-2 and heat shock proteins resulted in a redirection of metabolic fluxes. Catabolic pathways like the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid (TCA) cycle showed significantly enhanced activities, leading to reduced flux to growth-associated pathways like the pentose phosphate pathway and other anabolic pathways. Upon temperature upshift, an excess of NADPH was produced in the TCA cycle by isocitrate dehydrogenase. The metabolic model predicted the involvement of a transhydrogenase generating additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. The influence of the temperature upshift on the host's metabolism was investigated by means of a control strain harboring the "empty" parental expression vector. The metabolic fluxes after the temperature upshift were redirected similarly to the production strain; the effects, however, were observed to a lesser extent and with different time profiles.     

Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli

The kinetics of the heat-shock response and the formation of inclusion bodies in recombinant Escherichia coli TG1 were studied in glucose-limited high-cell-density cultures in response to temperature-induced production of human basic fibroblast growth factor (hFGF-2), a protein which partially aggregates into inclusion bodies. The maximum synthesis rates of heat-shock proteins were similar to those in a control cultivation with a strain carrying an expression vector without inducible structural gene. However, the maximum of induction for many heat-shock proteins including DnaK, ClpB, and HtpG was reached at least 30 min later when synthesis of hFGF-2 was simultaneously induced by the temperature upshift. During this first production phase, hFGF-2 was exclusively deposited in the insoluble cell fraction. Thereafter, accumulation of soluble hFGF-2 was observed, too, indicating that the recombinant protein needs heat-shock chaperones for proper folding at elevated temperatures. Strong recombinant protein production prolonged the synthesis of the majority of heat-shock proteins (including GroELS, DnaK, ClpB, and HtpG) even in a wildtype dnaK(+) background. In contrast, the synthesis rates of the small heat-shock proteins IbpA and IbpB declined within 1 h to preinduction values in control and hFGF-2 producing cultures. In the producing cultivation, IbpA and IbpB synthesis ceased to an undetectable level when soluble hFGF-2 started to accumulate, whereas the synthesis rates of the other heat-shock proteins including those belonging to the DnaK and GroEL families remained high throughout the entire production phase.     

Excretion of human beta-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli

Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage lambda to construct E. coli MBEL-BL101. For the excretion of human beta-endorphin as a model protein, the beta-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-beta-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-beta-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield beta-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of beta-endorphin was obtained.     

Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells

An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.     

Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation

Escherichia coli BL21 (DE3) and W3110 strains, belonging to the family B and K-12, respectively, have been most widely employed for recombinant protein production. During the excretory production of recombinant proteins by high cell density cultivation (HCDC) of these strains, other native E. coli proteins were also released. Thus, we analyzed the extracellular proteomes of E. coli BL21 (DE3) and W3110 during HCDC. E. coli BL21 (DE3) released more than twice the amount of protein compared with W3110 during HCDC. A total of 204 protein spots including 83 nonredundant proteins were unambiguously identified by 2-DE and MS. Of these, 32 proteins were conserved in the two strains, while 20 and 33 strain-specific proteins were identified for E. coli BL21 (DE3) and W3110, respectively. More than 70% of identified proteins were found to be of periplasmic origin. The outer membrane proteins, OmpA and OmpF, were most abundant. Two strains showed much different patterns in their released proteins. Also, cell density-dependent variations in the released proteins were observed in both strains. These findings summarized as reference proteome maps will be useful for studying protein release in further detail, and provide new strategies for enhanced excretory production of recombinant proteins.     

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.     

Kinetic model of in vivo folding and inclusion body formation in recombinant Escherichia coli

Aggregation of misfolded proteins can reduce the yield in recombinant protein production. The underlying complex processes are additionally influenced by cellular physiology. Nevertheless, a lumped-parameter model of kinetic competition between folding and aggregation was sufficient to track properly the specific concentration of a human protein produced in E. coli and its partitioning into soluble and insoluble cell fractions. Accurate estimation of the protein-specific parameters required informative experiments, which were designed using the Fisher information matrix. The model was employed to calculate the influence of the specific glucose uptake rate in high-cell-density cultivation of E. coli on accumulation and aggregation of the recombinant protein. Despite its simplicity, the model was flexible and unbiased concerning unidentified mechanisms. Assuming an exponentially decreasing production rate, the irreversible aggregation step was found to follow first order kinetics, while assuming a constant production rate with simultaneous degradation, the model predicted transient aggregation only. Implications for strain and process development are discussed.     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone l⁻¹ and 7 g fusion glucagon l⁻¹. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content. Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997     

Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions

Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.     

Recombinant protein secretion in Escherichia coli

The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost. This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.     

脯氨酰内肽酶培养条件的优化及高密度发酵

基因工程菌E.coliBL21/pGEMPEP可以组成型表达重组的点状产气单胞菌脯氨酰内肽酶(PEP),但培养条件极大地影响着酶的产量,为了获得高效表达,首先测定了工程菌表达PEP的稳定性并考察了培养温度、pH、发酵时间、碳源、氮源、无机盐等对产酶的影响,得到了优化的发酵条件,L9(3⁴)正交试验进一步明确了摇床转速、培养温度、pH值、培养时间对产酶量的影响都有高度的统计学意义。在此基础上利用NBSBioFlo3000型5L自控发酵罐进行了高密度、高表达发酵、经20h培养,最终菌体密度达OD₆₀₀60(相当于干菌体225g/L),PEP表达量为28%,每升发酵液中含PEP酶315g。     

Enhanced production of recombinant proteins in Escherichia coli by filamentation suppression

During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the E. coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.     

Analysis of factors affecting the periplasmic production of recombinant proteins in Escherichia coli

Five fusion proteins between Z domains derived from Staphylococcal Protein A and Green Fluorescent Protein or Human Proinsulin were produced on the periplasm of Escherichia coli. The effects of the molecular weight and amino acid composition of the translocated peptide, culture medium composition, and growth phase of the bacterial culture were analyzed regarding the expression and periplasmic secretion of the recombinant proteins. It was found that secretion was not affected by the size of the translocated peptide (17-42 kDa) and that the highest periplasmic production values were obtained on the exponential phase of growth. Moreover, the highest periplasmic values were obtained in minimal medium, showing the relevance of the culture medium composition on secretion. In silico prediction analysis suggested that with respect to the five proteins used in this study, those that are prone to form alpha-helix structures are more translocated to the periplasm.     

Extracellular release of recombinant alpha-amylase from Escherichia coli using pulsed electric field

It is difficult for Escherichia coli to secrete products such as recombinant enzymes, because the Gram-negative bacterium has a double membrane structure and so some of the products are accumulated in a periplasmic space. In this study, we demonstrated that recombinant alpha-amylase can be released from recombinant E. coli HB101/pHI301A during cultivation by applying a pulsed electric field (PEF). When a PEF (12 kV, 2 Hz) was applied for 30 min with an interval of 30 min from the point of OD660=0.7, the amount of released alpha-amylase was about 30% of the total amount of alpha-amylase produced in the cells. As a result of SDS-PAGE and activity staining analyses, it was confirmed that the released proteins were not all of the intracellular proteins, and the alpha-amylase, which was identical with intracellular alpha-amylase, was released by applied PEF cultivation. PEF treatment could be useful for easy release of periplasmic protein with selectivity.     

An approach to the production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli

The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ(32)). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by σ(32). One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of σ(32). The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes.