Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

Relationship of Escherichia coli density to growth rate and cell age.

The cell densities of Escherichia coli strains B/rA, BrF, and K-12 (OV-2) were measured at several growth rates and found to be very near 1.105 g/ml in all cases. Ninety percent of the cells of any exponentially growing population banded at densities differing less than 0.75% from the mean. Synchronized populations of B/rA selected as newborn cells were found to keep their density constant for longer than one generation time. However, if selection was based on cell size, by sedimentation through a sucrose gradient, cell density was found to be almost 2% lower than that of newborn cells, but it reached normal values before the first division had taken place. These results meant that mass and volume during the lifetime of the bacterial cell followed parallel kinetics. It was unlikely that density could regulate any event of the lifetime of a cell; on the contrary, density seemed to be a physical parameter that was well controlled during the bacterial growth.     

Independence of buoyant cell density and growth rate in Escherichia coli

The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.     

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.     

Fed-batch operation of recombinant Escherichia coli containing trp promoter with controlled specific growth rate

A feb-batch operation for the production of bovine somatotropin (bST) under the control of tryptophan promoter in Escherichia coli was investigated. The plasmid used contains a two-cistron system and altered codon usage for higher expression of bST. Specific growth rate is an important parameter in the fermentation, because it affects the production of growth-inhibitory organic acids and the expression of recombinant protein. The feeding rate was adjusted to keep the specific growth rate constant in this study. The variable growth yield expressed as a function of time was used for the calculation of the feeding rate. The growth yield decreases during the fermentation as product expression is induced. The specific growth rate was well controlled; however, intracellular bST concentration decreased at high cell concentrations. This is considered to be due to degradation by proteases. The decrease was prevented by an exponential feeding of the yeast extract as an organic nitrogen source. (c) 1994 John Wiley & Sons, Inc.     

High cell density cultivation of Escherichia coli W using sucrose as a carbon source

Summary Escherichia coli W was cultivated in the pH-stat fed-batch mode in a chemically defined medium containing sucrose as a sole carbon source. A final cell concentration of 105 g/L could be obtained in 36 hrs by feeding 20 g sucrose+0.57 g MgSO₄7H₂O for a definite on-time upon the pH rise. Using the same strategy, a recombinant strain harboring pSYL104 could also be grown to 125 g/L in 48 hrs.     

High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production

Glucagon-like peptide-1 (GLP-1)(2) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.     

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.     

Simple fed-batch technique for high cell density cultivation of Escherichia coli

A simple fed-batch process for high cell density cultivation of Escherichia coli TG1 was developed. A pre-determined feeding strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor controlling biomass accumulation at growth rates which do not cause the formation of acetic acid (μ < μ_crit). Cell concentrations of 128 and 148 g per 1 dry cell weight (g 1⁻¹ DCW) were obtained using glucose or glycerol as carbon source, respectively.     

Estimation of specific growth rate from cell density measurements

For modelling purposes it is of great importance to derive the specific growth rate as a function of time from biomass measurements. Traditional methods such as exponential or polynomial fitting do not give satisfactory results nor do these methods take the noise characteristics of the biomass measurements into account. Standard recursive techniques, such as Kalman filtering, use only the data up to the time under consideration and are dependent of a good initial estimation. This paper describes a technique based on combining subsequent backward and forward extended Kalman filtering to give a smoothing estimator for the specific growth rate. The estimator does not need an initial value and is shown to have a single tuning parameter. The applicability of the estimator is demonstrated on batch and fed-batch cultivations of two organisms: Bordetella pertussis and Neisseria meningitidis.     

Evidence for osmoregulation of cell growth and buoyant density in Escherichia coli.

The buoyant density of cells of Escherichia coli B/r NC32 increased with the osmolarity of the growth medium. Growth rate and its variability were also dependent upon the osmolarity of the medium. Maximum growth rates and minimum variability of these rates were obtained in Luria broth by addition of NaCl to a concentration of about 0.23 M.     

Bilinear cell growth of Escherichia coli.

Recent electron micrograph measurements of bacterial dimensions in exponentially growing cultures of Escherichia coli support a model of bilinear increase in cell surface area and volume, with a sharp doubling in growth rate at a discrete age during the cell cycle. The results also indicate coordinate regulation of increase of surface area and volume.     

Generic model control of the specific growth rate in recombinant Escherichia coli cultivations

Generic model control is shown to be a powerful tool for keeping a microbial cultivation process close to its predetermined (optimized) control profile. This is demonstrated at the example of the green fluorescent protein expressed in genetically modified Escherichia coli host cells. It is shown that the process can be run very closely to a predefined complex profile of the specific cell growth rate mu(t). Controlling the experiments at many different growth conditions is a straightforward way of effectively collecting the data necessary for optimization of recombinant protein production systems. Although the process dynamics is rather complex, the model for the controller can be kept quite simple. The control technique, used here for specific growth rate control, is quite universal and can be applied for different biotechnological processes as well.     

High-cell-density cultivation of Escherichia coli.

High-cell-density cultivations of Escherichia coli in glucose-mineral-salt media produce more than 100 g dry cells litre-1 in special fed-batch modes with feeding of glucose and ammonia only. The specific growth rate can be adjusted to allow optimum recombinant protein generation.     

Fuzzy neural network for the control of high cell density cultivation of recombinant Escherichia coli

A five-layer fuzzy neural network (FNN) was developed for the control of fed-batch cultivation of recombinant Escherichia coli JM103 harboring plasmid pUR 2921. The FNN was believed to represent the membership functions of the fuzzy subsets and to implement fuzzy inference using previous experimental data. This FNN was then used for compensating the exponential feeding rate determined by the feedforward control element. The control system is therefore a feedforward-feedback type. The change in pH of the culture broth and the specific growth rate were used as the inputs to FNN to calculate the glucose feeding rate. A cell density of 84 g DWC/l in the fed-batch cultivation of the recombinant E. coli was obtained with this control strategy. Two different FNNs were then employed before and after induction to enhance plasmid-encoded β-galactosidase production. Before induction the specific growth rate was set as 0.31 h⁻¹, while it was changed to 0.1 h⁻¹ after induction. Compared to when only one FNN was used, the residual glucose concentration could be tightly controlled at an appropriate level by employing two FNNs, resulting in an increase in relative activity of β-galactosidase which was about four times greater. The present investigation demonstrates that a feedforward-feedback control strategy with FNN is a promising control strategy for the control of high cell density cultivation and high expression of a target gene in fed-batch cultivation of a recombinant strain.     

RelE-mediated dormancy is enhanced at high cell density in Escherichia coli

Bacteria show remarkable adaptability under several stressful conditions by shifting themselves into a dormant state. Less is known, however, about the mechanism underlying the cell transition to dormancy. Here, we report that the transition to dormant states is mediated by one of the major toxin-antitoxin systems, RelEB, in a cell density-dependent manner in Escherichia coli K-12 MG1655. We constructed a strain, IKA121, which expresses the toxin RelE in the presence of rhamnose and lacks chromosomal relBE and rhaBAD. With this strain, we demonstrated that RelE-mediated dormancy is enhanced at high cell densities compared to that at low cell densities. The initiation of expression of the antitoxin RelB from a plasmid, pCA24N, reversed RelE-mediated dormancy in bacterial cultures. The activation of RelE increased the appearance of persister cells against β-lactams, quinolones, and aminoglycosides, and more persister cells appeared at high cell densities than at low cell densities. Further analysis indicated that amino acid starvation and an uncharacterized extracellular heat-labile substance promote RelE-mediated dormancy. This is a first report on the induction of RelE-mediated dormancy by high cell density. This work establishes a population-based dormancy mechanism to help explain E. coli survival in stressful environments.     

Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli.

The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography. At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1). The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly. Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material. In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall.     

高密度培养重组大肠杆菌发酵生产胆固醇氧化酶的策略

以重组大肠杆菌发酵生产胆固醇氧化酶,依据溶解氧的变化控制底物的流加速率,实现了重组大肠杆菌的高密度培养,最高密度达85（OD600）。在此基础上确定了最佳的诱导时机为发酵中期,菌体产酶水平达5830．6tJ／L,产酶速率为971．77U·L^-1·h^-1,生产强度为388．71U·L^-1·h^-1,实现了胆固醇氧化酶的高效生产。