Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production

The culture of Escherichia coli for the commercial production of recombinant proteins has increased significantly in recent years. The production of acetate as a byproduct retards cell growth, inhibits protein formation, and diverts carbon from biomass to protein product. Our approach to reducing acetate accumulation was to disable the phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) by deleting the ptsHI operon in the wild-type E. coli strain GJT001. The mutation caused a severe reduction in growth rate and glucose uptake rate in glucose-supplemented M9 minimal medium, which confirmed the mutation, and eliminated acetate accumulation. The mutant strain (TC110) apparently metabolized glucose by a non-PTS mechanism that we are currently investigating, followed by phosphorylation by glucokinase. In complex medium such as 2xLB broth with 2% glucose, TC110 was able to grow quickly and still retained the phenotype of significantly reduced acetate accumulation (9.1+/-6.6 vs. 90.4+/-1.6mM in GJT001, P<0.05). The reduced acetate accumulation resulted in a significant improvement in final OD (23.5+/-0.7 in TC110 vs. 8.0+/-0.1 in GJT001, P<0.05). We tested the strains for the production of model recombinant proteins such as green fluorescent protein (GFP) and beta-galactosidase. TC110 had a 385-fold improvement in final volumetric productivity of GFP over GJT001 in shake flasks with 2xLB broth with 2% glucose. The distribution of GFP fluorescence in the cell population, as determined by flow cytometry, was much broader in GJT001 (coefficient of variation=466+/-35%) than in TC110 (coefficient of variation=55+/-1%). In corn steep liquor medium with 2% glucose, we observed a 28.5-fold improvement in final volumetric production of GFP in TC110 over GJT001. TC110 had a 7.5-fold improvement in final volumetric productivity of beta-galactosidase over GJT001 in 2xLB broth with 2% glucose medium. When tested in a batch bioreactor cultures with 2xLB broth with 2% glucose medium, the volumetric production of GFP by TC110 was 25-fold higher than that of GJT001. In summary, the ptsHI mutant of GJT001 resulted in reduced acetate accumulation, which led to significant improvements in recombinant protein production in batch bioreactors.     

Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations

Metabolic footprinting has become a valuable analytical approach for the characterization of phenotypes and the distinction of specific metabolic states resulting from environmental and/or genetic alterations. The metabolic impact of heterologous protein production in Escherichia coli cells is of particular interest, since there are numerous cellular stresses triggered during this process that limit the overall productivity, e.g. the stringent response. Because the knowledge on the metabolic responses in recombinant bioprocesses is still scarce, metabolic footprinting can provide relevant information on the intrinsic metabolic adjustments. Thus, the metabolic footprints generated by E. coli W3110 and the ΔrelA mutant strain during recombinant fed-batch fermentations at different experimental conditions were measured and interpreted. The IPTG-induction of the heterologous protein expression resulted in the rapid accumulation of inhibitors of the glyoxylate shunt in the culture broth, suggesting the clearance of this anaplerotic route to replenish the TCA intermediaries withdrawn for the additional formation of the heterologous protein. Nutritional shifts were also critical in the recombinant cellular metabolism, indicating that cells employ diverse strategies to counteract imbalances in the cellular metabolism, including the secretion of certain metabolites that are, most likely, used as a metabolic relief to survival processes.     

Use of a stress-minimisation paradigm in high cell density fed-batch Escherichia coli fermentations to optimise recombinant protein production

Production of recombinant proteins is an industrially important technique in the biopharmaceutical sector. Many recombinant proteins are problematic to generate in a soluble form in bacteria as they readily form insoluble inclusion bodies. Recombinant protein solubility can be enhanced by minimising stress imposed on bacteria through decreasing growth temperature and the rate of recombinant protein production. In this study, we determined whether these stress-minimisation techniques can be successfully applied to industrially relevant high cell density Escherichia coli fermentations generating a recombinant protein prone to forming inclusion bodies, CheY–GFP. Flow cytometry was used as a routine technique to rapidly determine bacterial productivity and physiology at the single cell level, enabling determination of culture heterogeneity. We show that stress minimisation can be applied to high cell density fermentations (up to a dry cell weight of >70 g L^?1) using semi-defined media and glucose or glycerol as carbon sources, and using early or late induction of recombinant protein production, to produce high yields (up to 6 g L^?1) of aggregation-prone recombinant protein in a soluble form. These results clearly demonstrate that stress minimisation is a viable option for the optimisation of high cell density industrial fermentations for the production of high yields of difficult-to-produce recombinant proteins, and present a workflow for the application of stress-minimisation techniques in a variety of fermentation protocols.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Control of ammonium concentration in Escherichia coli fermentations

A control system has been devised for the maintenance of stable ammonium concentrations throughout a fedbatch fermentation. The control system is based on an ammonia gas-sensing electrode that monitors a pH-adjusted effluent stream from the fermentor. To overcome the time lag between the fermentor and the electrode, feedback control included metered flows of ammonium to both the fermentor and the electrode vessel. The system was used to study the growth of Escherichia coli B (ATCC 11303) at controlled ammonium concentrations of 5 to 200mM. Apparent specific growth rates, biomass and protein production, and glucose yields were essentially constant from 5 to 170mM. Above 170mM ammonium growth was inhibited. As ammonium concentration decreased from 170 to 5mM, ammonium yields increased from 1 to 24 g cell dry wt/g ammonium utilized. The results demonstrate that control of ammonium concentrations at levels so low that ammonium would be exhausted in batch fermentations can significantly increase overall ammonium yields.     

Maintaining rapid growth in moderate-density Escherichia coli fermentations

A novel feeding strategy that prolongs rapid growth rates for Escherichia coli fermentations to moderately high cell density is presented. High-density fermentations are a common and successful means of producing biological products. However, acetate accumulation can be a substantial problem in these procedures. To avoid this problem, many feeding strategies and host modifications have been developed, but all result in relatively low growth rates. If a faster growth rate could be maintained, the growth phase of the process would be shortened, leading to increased productivity. It is also possible that the subsequent specific production rate could be enhanced by growing the early culture at a faster rate. We have developed a procedure to enable rapid growth to a cell density of 20 g/L and have used cell-free protein synthesis to evaluate the relative potential of the resulting cells for producing recombinant proteins. The method uses glucose pulses and the duration of the dissolved oxygen response to calculate the appropriate glucose feed rate based on the glucose demand of the culture. Amino acids and vitamins were supplied in the medium to increase the growth rate. We were able to sustain a growth rate of 0.8/h up to 20 g/L dry cell weight without significant acetate accumulation. Analysis of amino acid consumption indicates that cell composition is an accurate predictor of amino acid demand for most amino acids. Cell-free protein synthesis was used to compare the protein production potential of the high-density cultures with that of cells grown in complex medium and harvested at low cell density and maximum growth rate. Protein production for the extract from the controlled, high-density fermentations was 950 mg/L compared with 860 mg/L for the low-density control. Therefore, the new control procedure has promising potential for developing rapid and productive industrial fermentations.     

Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.     

Automatic containment sampling of recombinant escherichia coli fermentations

A containment sampling system for shake flasks and fermentors has been developed from a blood collection system used in hospitals. The core of the system is a collection vial with a vacuum inside. When a needle connected to the fermentation fluid penetrates a rubber seal on the vial, a sample is withdrawn. The system has been developed in two versions, a manual method for shake flasks, and an automated version for fermentors including cool storage of samples. The sampling system offers the same safety for fermentation containment as the original system offers safety for patients and hospital staff. (c) 1992 John Wiley & Sons, Inc.     

The potential of random forest and neural networks for biomass and recombinant protein modeling in Escherichia coli fed‐batch fermentations

Product quality assurance strategies in production of biopharmaceuticals currently undergo a transformation from empirical “quality by testing” to rational, knowledge‐based “quality by design” approaches. The major challenges in this context are the fragmentary understanding of bioprocesses and the severely limited real‐time access to process variables related to product quality and quantity. Data driven modeling of process variables in combination with model predictive process control concepts represent a potential solution to these problems. The selection of statistical techniques best qualified for bioprocess data analysis and modeling is a key criterion. In this work a series of recombinant Escherichia coli fed‐batch production processes with varying cultivation conditions employing a comprehensive on‐ and offline process monitoring platform was conducted. The applicability of two machine learning methods, random forest and neural networks, for the prediction of cell dry mass and recombinant protein based on online available process parameters and two‐dimensional multi‐wavelength fluorescence spectroscopy is investigated. Models solely based on routinely measured process variables give a satisfying prediction accuracy of about ± 4% for the cell dry mass, while additional spectroscopic information allows for an estimation of the protein concentration within ± 12%. The results clearly argue for a combined approach: neural networks as modeling technique and random forest as variable selection tool.     

Expanding the landscape of recombinant protein production in Escherichia coli

Over the years, several vectors and host strains have been constructed to improve the overexpression of recombinant proteins in Escherichia coli. More recently, attention has focused on the co-expression of genes in E. coli, either by means of a single vector or by cotransformation with multiple compatible plasmids. Co-expression was initially designed to generate protein complexes in vivo, and later served to extend the use of E. coli as a platform for the production of heterologous proteins. This review shows how the co-expression of genes in E. coli is challenging the production of protein complexes and proteins bearing post-translational modifications or unnatural amino acids. In addition, the importance of co-expression to achieve efficient secretion of recombinant proteins in E. coli is discussed, with recent insights into the use of co-expression to overproduce membrane proteins.     

Metabolic pathway structures for recombinant protein synthesis in Escherichia coli

Escherichia coli is a valuable commercial host for the production of heterologous proteins. We used elementary mode analysis to identify all possible genetically independent pathways for the production of three specific recombinant proteins, green fluorescent protein, savinase and an artificial protein consisting of repeating units of a five-amino-acid cassette. Analysis of these pathways led to the identification of the most efficient pathways for the production of each of these proteins. The results indicate that the amino acid composition of expressed proteins has a profound effect on the number and identity of possible pathways for the production of these proteins. We show that several groups of elementary modes produce the same ratio of biomass and recombinant protein. The pattern of occurrence of these modes is dependent on the amino acid composition of the specific foreign protein produced. These pathways are formed as systemic combinations of other pathways that produce biomass or foreign protein alone after the elimination of fluxes in specific internal reversible reactions or the reversible carbon dioxide exchange reaction. Since these modes represent pathway options that enable the cell to produce biomass and protein without utilizing these reactions, removal of these reactions would constrain the cells to utilize these modes for producing biomass and foreign protein at constant ratios.     

Advanced genetic strategies for recombinant protein expression in Escherichia coli

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology. Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E. coli cytoplasm. Here we review the current most important strategies for recombinant expression in E. coli. Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology. The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.     

Elevated Fis expression enhances recombinant protein production in Escherichia coli

A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138-144, 1997.     

Self-cycling operation increases productivity of recombinant protein in Escherichia coli

Self-cycling fermentation (SCF), a cyclical, semi-continuous process that induces cell synchrony, was incorporated into a recombinant protein production scheme. Escherichia coli CY15050, a lac(-) mutant lysogenized with temperature-sensitive phage λ modified to over-express β-galactosidase, was used as a model system. The production scheme was divided into two de-coupled stages. The host cells were cultured under SCF operation in the first stage before being brought to a second stage where protein production was induced. In the first stage, the host strain demonstrated a stable cycling pattern immediately following the first cycle. This reproducible pattern was maintained over the course of the experiments and a significant degree of cell synchrony was obtained. By growing cells using SCF, productivity increased 50% and production time decreased by 40% compared to a batch culture under similar conditions. In addition, synchronized cultures induced from the end of a SCF cycle displayed shorter lysis times and a more complete culture-wide lysis than unsynchronized cultures. Finally, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle. For example, induction of a synchronized culture immediately prior to cell division resulted in the maximum protein productivity, suggesting protein production can be optimized with respect to the cell life cycle using SCF.     

大肠杆菌高效表达重组蛋白策略

大肠杆菌表达系统是基因表达技术中发展最早和目前应用最广的经典表达系统。利用该系统表达重组蛋白具有许多优越性,但其表达效率受诸多因素的影响。本文综述国内外利用大肠杆菌表达系统高效表达外源蛋白的策略,主要包括选择合适的启动子、改变信号肽结构、提高mRNA稳定性、提高翻译效率、表达稀有密码子、降低包涵体形成及蛋白降解,利用融合蛋白与分子伴侣、调控发酵条件实现高密度培养等。     

Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.