Macromolecule synthesis of Escherichia coli BB at a lower or transient growth state.

Macromolecule synthesis in Escherichia coli BB at lower growth rates was investigated. The results indicate that a deviation in ribonucleic acid (RNA) content per cell at a lower growth rate from the exponential relationship to a specific growth rate is entirely attributable to the presence of nonviable cells, in which the RNA content is lower than in viable cells. Based on this fact, a mathematical expression of macromolecule contents versus specific growth rate was devised. Moreover, continuous changes in macromolecule content during unbalanced growth from late-logarithmic phase to stationary phase were measured. Although growth rates changed continuously, the data on deoxyribonucleic acid (DNA) or RNA content versus the specific growth rate calculated from the increments in cell number satisfactorily fitted the exponential lines obtained under balanced growth at a higher growth rate. However, no such relationship was observed in the plot of DNA or RNA content versus the specific growth rate calculated from the increments in optical density.     

Macromolecule synthesis of Escherichia coli BB at a lower or transient growth state.

Macromolecule synthesis in Escherichia coli BB at lower growth rates was investigated. The results indicate that a deviation in ribonucleic acid (RNA) content per cell at a lower growth rate from the exponential relationship to a specific growth rate is entirely attributable to the presence of nonviable cells, in which the RNA content is lower than in viable cells. Based on this fact, a mathematical expression of macromolecule contents versus specific growth rate was devised. Moreover, continuous changes in macromolecule content during unbalanced growth from late-logarithmic phase to stationary phase were measured. Although growth rates changed continuously, the data on deoxyribonucleic acid (DNA) or RNA content versus the specific growth rate calculated from the increments in cell number satisfactorily fitted the exponential lines obtained under balanced growth at a higher growth rate. However, no such relationship was observed in the plot of DNA or RNA content versus the specific growth rate calculated from the increments in optical density.     

Reducing the glucose uptake rate in Escherichia coli affects growth rate but not protein production

Although glucose is an inexpensive substrate widely used as a carbon source in Escherichia coli recombinant fermentation technology, 10-30% of the carbon supply is wasted by excreting acetate. In addition to the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. Because glucose can enter the cell via several transport systems, isogenic strains defective in one or two of these transport systems were constructed. The effects of changes in the glucose uptake capacity on the in vivo flux distribution to a desired end product (beta-galactosidase) and to acetate were studied. The lack of one of the components (IICB(Glc) protein) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced the growth rate significantly. The maintenance of a low-copy plasmid in this strain resulted in further arrest of the growth rate. However, beta-galactosidase production had no effect on growth rate. This strain directed more carbon into biomass and carbon dioxide, and less into acetate. Beta-galactosidase was produced in amounts not significantly different from the wild-type strain from half the amount of glucose. An explanation for the experimental results is given, making use of published results on metabolic regulation.     

Macromolecule synthesis in Escherichia coli BB under various growth conditions.

The kinetic behavior of the macromolecule synthesis of Escherichia coli during balanced growth in various media at different temperatures as investigated. The results indicate that macromolecule contents per cell can be expressed as exponential functions of the specific growth rate at a given temperature. It was shown that the content per cell at the zero growth rate was constant in each macromolecule component, irrespective of the growth temperature. The rate of ribonucleic acid (RNA) synthesis per unit weight of deoxyribonucleic acid and that of protein synthesis per unit weight of RNA were taken as efficiencies of RNA and protein synthesis, respectively; both of them were found to be dependent on the growth rate and temperature. The efficiency of RNA synthesis was found to be very high at a high growth rate, whereas that of protein synthesis was found to decrease above certain growth rate. At the same growth rate, an increase in the growth temperature resulted in a decrease in the efficiency of RNA synthesis but an increase in that of protein synthesis.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Dependence of the specific growth rate of Escherichia coli MI 30 on the ammonia concentration]

The transient behaviour of ammonium limited continuous cultures of E. coli ML 30 led to the hypothesis that the bistability of pyruvate formation primarily is caused by a bistability of the ammonia metabolism. Therefore, a function of mu([NH+4]) should be expected different from that of Monod type. Measurements of the specific growth rate during washout of continuous cultures at different ammonium concentrations and at such low cell concentrations that the changes in the ammonium concentration of the medium could be neglected, showed a complex function with a relative minimum near 2 mg/1NH+4. This function allows bistability of the ammonium concentration in an ammonium limited continuous culture. The results are discussed on the basis of the two systems of ammonia assimilation found in prokaryotic cells.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Pilot scale exponential growth of Escherichia coli W to high cell concentration with temperature variation

An efficient method to grow Escherichia coli W to high cell concentrations on the pilot scale is described and discussed. The method involves growth linked introduction of glucose; and ammonia to the culture, sparing with oxygen, and maintenance of aerobic conditions by gradually decreasing the temperature in the culture in order to keep the oxygen demand within the limits of the capacity of supply. Under these conditions the linear rate of cell mass production is actually the result of exponential growth with a gradually decreasing growth-rate constant. About 10 kg packed cells were produced in a 50 liter working-volume fermentor in one run of 13 hr. The concentration of the cells at the end of the growth was about 47 g dry cells/liter. The expenditure for nutrients was minimal and the controls were of simple automatic nature. From the determined yield constants for glucose, nitrogen, phosphorus, and oxygen it may be inferred that the cells grown by this method are similar to those grown exponentially at constant temperature.     

High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.     

BipA is required for growth of Escherichia coli K12 at low temperature

The bipA gene encodes a ribosome-associated GTPase postulated to be involved in regulatory functions in enteropathogenic Escherichia coli. Previous studies demonstrated that BipA is tyrosine phosphorylated in EPEC strains, but not in E. coli strain K12. Results presented here indicate that BipA function is required at low temperatures in E. coli K12, suggesting a regulatory role independent of phosphorylation and of pathogenicity.     

Mutation of Thr445 and Ile500 of initiation factor 2 G-domain affects Escherichia coli growth rate at low temperature

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.     

Isopycnography of intact cells - III: concentration of ribosomes in Escherichia coli versus growth rate

The relative quantification of the ribosomal content in a population of cells permits correlation between growth rate and available protein synthesis machinery. Isopycnometry of living cells indicates that this correlation is linear in $\text{E.}\text{coli}$ B. Additionally, the narrow band-widths imply great homogeneity of response by the population at any one time. The precision of this method appears to surpass that of previous determinations.     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h⁻¹ in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.     

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.     

Macromolecule Synthesis of Escherichia coli BB at a Lower or Transient Growth State

[This corrects the article on p. 751 in vol. 34.].