Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions

Three E. coli strains, named VAL22, VAL23, and VAL24, were engineered at the level of mixed-acid fermentation pathways to improve culture performance under transient anaerobic conditions. VAL22 is a single mutant with an inactivated poxB gene that codes for pyruvate oxidase which converts pyruvate to acetate. VAL23 is a double mutant unable to produce lactate and formate due to deletions of the ldhA and pflB genes that code for lactate dehydrogenase and pyruvate-formate lyase, respectively. VAL24 is a triple mutant with ldhA and pflB deleted and poxB inactivated. Engineered strains were cultured under oscillating dissolved oxygen tension (DOT) in a scale-down system, to simulate gradients occurring in large-scale bioreactors. Kinetic and stoichiometric parameters of constant (10%) and oscillating DOT cultures of the engineered strains were compared with those of the parental strain, W3110. All strains expressed recombinant green fluorescent protein (GFP) as a protein model. Mutant strains showed improved specific growth rate, reduced by-product formation, and reduced specific glucose uptake rate compared to the parental strain, when cultured under oscillating DOT. In particular, lactate and formate production was abolished and acetate accumulation was reduced by 9-12%s. VAL24 showed the best performance, as specific growth and GFP production rates, and maximum GFP concentration were not affected by DOT gradients and were at least twofold higher than those of W3110 under constant DOT. Under oscillating DOT, VAL24 wasted about 40% less carbon into fermentation by-products than W3110. It was demonstrated that, although E. coli responds rapidly to DOT fluctuations by deviating to fermentative metabolism, such pathways can be eliminated as they are not necessary for bacterial survival during the short circulation times typical of large-scale cultures. The approach shown here opens new possibilities for designing metabolically engineered strains, with reduced sensitivity to DOT gradients and improved performance under typical conditions of large-scale cultures.     

Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system: metabolic response and production of recombinant protein

A significant problem of large-scale cultures, but scarcely studied for recombinant E. coli, is the presence of gradients in dissolved oxygen tension (DOT). In this study, the effect of DOT gradients on the metabolic response of E. coli and production of recombinant pre-proinsulin, accumulated as inclusion bodies, was determined. DOT gradients were simulated in a two-compartment scale-down system consisting of two interconnected stirred-tank bioreactors, one maintained at anoxic conditions and the other at a DOT of at least 6%. Cells were continuously circulated between both vessels to simulate circulation times (tc) of 20, 50, 90, and 180 sec. A complete kinetic and stoichiometric characterization was performed in the scale-down system as well as in control cultures maintained at constant DOT in the range of 0-20%. The performance of E. coli cultured under oscillating DOT was significantly affected, even at a tc of 20 sec corresponding to transient exposures of only 13.3 sec to anaerobic conditions. Specific growth rate decreased linearly with tc to a maximum reduction of 30% at the highest tc tested. The negative effect of DOT gradients was even more pronounced for the overall biomass yield on glucose and the maximum concentration and yield of pre-proinsulin. In these cases, the losses were 9%, 27%, and 20%, respectively, at tc of 20 sec and 65%, 94%, and 87%, respectively, at tc of 180 sec. Acetic, lactic, formic, and succinic acids accumulated during oscillatory DOT cultures, indicating that deviation of carbon flow to anaerobic metabolism was responsible for the observed losses. The results of this study indicate that even very short exposures to anaerobic conditions, typical of large-scale operations, can substantially reduce recombinant protein productivity. The information presented here is useful for establishing improved rational scale-up strategies and understanding the behavior of recombinant E. coli exposed to DOT gradients.     

Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii.

The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced. The higher the amplitude, the lower the MMW(max). As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.     

Control of redox potential in hybridoma cultures: effects on MAb production, metabolism, and apoptosis

Culture redox potential (CRP) has proven to be a valuable monitoring tool in several areas of biotechnology; however, it has been scarcely used in animal cell culture. In this work, a proportional feedback control was employed, for the first time, to maintain the CRP at different constant values in hybridoma batch cultures for production of a monoclonal antibody (MAb). Reducing and oxidant conditions, in the range of -130 and +70 mV, were maintained in 1-l bioreactors through automatic control of the inlet gas composition. Cultures at constant DOT, in the range of 3 and 300 %, were used for comparison. The effect of constant CRP on cell concentration, MAb production, metabolism of glucose, glutamine, thiols, oxygen consumption, and programmed cell death, was evaluated. Reducing conditions resulted in the highest viable cell and MAb concentrations and thiols production, whereas specific glucose and glutamine consumption rates remained at the lowest values. In such conditions, programmed cell death, particularly apoptosis, occurred only after nutrient exhaustion. The optimum specific MAb production rate occurred at intermediate CRP levels. Oxidant conditions resulted in a detrimental effect in all culture parameters, increasing the specific glucose, glutamine, and oxygen consumption rates and inducing the apoptotic process, which was detected as early as 24 h even when glutamine and glucose were present at non-limiting concentrations. In most cases, such results were similar to those obtained in control cultures at constant DOT.     

In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse‐flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse‐flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non‐feeding at a dilution rate of 0.2 h^?1 results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short‐term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi‐continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h^?1 and 0.5 h^?1, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h^?1 – 0.5 h^?1. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1348–1355, 2014     

Quasi-continuous fermentation in a reverse-flow diafiltration bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse-flow diafiltration utilizes an internal membrane device, but pulsed feeding causes spatial heterogeneities. In this study, the influence of conventional reverse-flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non-feeding at a dilution rate of 0.2 h⁻¹ results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short-term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi-continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h⁻¹ and 0.5 h⁻¹, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h⁻¹ to 0.5 h⁻¹. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time.     

Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of Azotobacter vinelandii*

The alginate production by Azotobacter vinelandii, as well as the molecular weight of the polymer, are strongly influenced by the dissolved oxygen tension (DOT) and stirring speed of the culture. Under high DOT (5% of air saturation), the bacteria produced more alginate (4.5 g/l) than that obtained at low (0.5%) oxygen tension (1.0 g/l) in cultures conducted at 300 rpm. On the other hand, under constant DOT (3%), the higher the stirring speed (from 300 to 700 rev./min), the higher the specific growth rate and the alginate production rate. However, low agitation speed (300 rev./min) lead the culture to produce a polymer of high molecular weight (680 000 g/g mol) whereas a low molecular weight (352 000 g/g mol) alginate was isolated from cultures conducted at high (700 rev./min) stirring speed. At 700 rev./min, the MMW increased to a plateau between 1 and 3% DOT and then decreased to a minimum of 0.11 x 10(6) g/g mol at 7%. Microscopic observations revealed the presence of cell aggregates (one order of magnitude larger than individual cells) when the culture was conducted at 300 rev./min. Oxygen gradients occurring within the aggregates could be responsible of this phenomenon. At high agitation rate, the MMW of the alginate dropped towards the end of the culture in all conditions evaluated. Alginase activity was detected, which would be responsible for this phenomenon.     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

Media for hybridoma growth and monoclonal antibody production

For the economical production of monoclonal antibodies (MAbs), the cell-culture medium must be optimized for three different phases: growth of the hybridomas, MAb productivity of the hybridomas, and MAb purification or downstream processing. Medium improvements are necessary to meet these requirements for large-scale MAb production. Information bearing on this issue is being addressed in two research areas, cell biology and biochemical engineering, and is reviewed in this article.     

Effects of high cell density on growth-associated monoclonal antibody production by hybridoma T0405 cells immobilized in macroporous cellulose carriers

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production of hybridoma T0405 cells immobilized in macroporous cellulose carriers were investigated in continuous and batch cultures. The results showing, that the specific MAb production rate increased with increasing specific growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. Moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomena, MAb mRNA expression and cell cycle distribution were investigated in batch cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromode-oxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb mRNA expression reached the peak during the exponential growth phase, suggest a positively growth-associated MAb production. And the immobilized cells continued the MAb mRNA expression until dead phase, which was longer than that in suspended cells. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated MAb productivity of T0405 cells.     

Frequency analysis of autonomously oscillating yeast cultures

Measurements of state variables from oscillating chemostat cultures of Saccharomyces cerevisiae were analyzed by Fourier transformation. Of the signals tested, carbon dioxide and oxygen in the exit gas stream and dissolved oxygen in the medium, all gave identical results. Analysis of data from reactors operated at fixed conditions showed that after oscillations start, they pass through an extended transient lasting several days, before the oscillation period becomes constant. Under transient operating conditions, Fourier analysis revealed expected qualitative trends in the change of oscillation period with dilution rate.     

Osteoblastic cells have refractory periods for fluid-flow-induced intracellular calcium oscillations for short bouts of flow and display multiple low-magnitude oscillations during long-term flow

Partitioning a daily mechanical stimulus into discrete loading bouts enhances bone formation in rat tibiae (J. Bone Mineral Res. 15(8) (2000) 1596). We hypothesized that a refractory period exists in primary rat osteoblastic cells, during which fluid-flow-induced [Ca(2+)](i) oscillations are insensitive to additional short bouts (2 min) of fluid flow. Because the frequency of [Ca(2+)](i) oscillations is believed to be important for regulating cellular activity and long-term fluid flow alters gene expression in bone cells, we also hypothesized that long-term (15 min) oscillating fluid flow produces multiple [Ca(2+)](i) oscillations in osteoblastic cells. Primary osteoblastic cells from rat long bones were exposed to 2 min of oscillating fluid flow that produced shear stresses of 2 Pa at 2 Hz. After a rest period of 5, 30, 60, 300, 600, 900, 1800, or 2700 s, the cells were exposed to a second 2-min bout of flow. A 600 s rest period was required to recover the percentage of cells responding to fluid flow and a 900 s rest period was required to recover the [Ca(2+)](i) oscillation magnitude. The magnitude and shape of the two [Ca(2+)](i) oscillations were strikingly similar for individual cells after a 900 s rest period. During 15 min of continuous oscillating flow, some individual cells displayed between 1 and 9 oscillations subsequent to the initial [Ca(2+)](i) oscillation. However, only 54% of the cells that responded initially displayed subsequent [Ca(2+)](i) oscillations during long-term flow and the magnitude of the subsequent oscillations was only 28% of the initial response.     

The influence of experimentally generated turbulence on the Mash01 unicellular Microcystis aeruginosa strain

Phytoplankton experience a continuously changing fluid environment and the response to this is reflected at individual and community levels. The large-scale motions of winds, waves and artificial circulations are coupled by turbulence to the viscous small-scale environment of the phytoplankton cell. To investigate the significance of turbulence in the ecology of Microcystis aeruginosa, cultures were exposed to turbulent conditions using a vertically oscillating grid for a period of 7 days under controlled laboratory conditions. M. aeruginosa was exposed to a range of turbulent intensities, by adjusting the frequency of oscillation from 1 to 4 Hz. To improve the resolution of scale between turbulence phenomena and phytoplankton, flow cytometry and fluorescent probes were used to assess the response of M. aeruginosa. Metabolic activity and cell viability were monitored daily in both the turbulent cultures and quiescent control cultures using the FDA and Sytox green fluorescent probes, respectively. Initially, low turbulence levels generated by the grid at frequencies of 1 and 2 Hz stimulated metabolic activity, and did not affect cell viability compared to the control quiescent cultures. However, higher levels of turbulence generated by the grid at frequencies of 3 and 4 Hz were deleterious to metabolic activity and viability. Metabolic activity significantly decreased and over 85 % of cells were nonviable after 96 h at a grid oscillation of 4 Hz. It was concluded that due to the long lag time (>96 h) and high intensities needed to exert a deleterious effect, small-scale turbulence is unlikely to be a significant factor controlling M. aeruginosa compared to large scale motion which lead to changes in light and nutrient conditions.     

Continuous photoautotrophic cultures of the eukaryotic alga Chlorella vulgaris can exhibit stable oscillatory dynamics

Sustained oscillations in cell concentration, average per cell DNA content, and average cell size were found in continuous photoautotrophic cultures of Chlorella vulgaris at low dilution rates (0.1/day). The period of oscillation was approximately 10 days. DNA histograms determined by flow cytometry exhibited reproducible pattern through consecutive oscillations. At the maximum cell concentration during an oscillation, the DNA histograms showed that the majority of the cells were not replicating their chromosomes, and most of the culture was comprised of single cells in G0/G1 phase. The cells then initiated DNA replication; however, because of the long generation time, the cell concentration decreased to a minimum, and at the same time the average per cell DNA content reached its maximum value. At this point the cells began to divide, and the cell concentration increased until it reached its maximum value at the beginning of the next oscillation. Calculations based on the supplied nutrients and comparison to biomass generation showed that the oscillatory behavior in continuous photoautotrophic cultures of C. vulgaris was not due to nutrient limitation, but most likely was due to the secretion of compounds that alter cell cycle kinetics. The oscillatory behavior disappeared when the dilution rate was increased to 0.3/day and the culture reached a stable steady state.     

Bioluminescent monitoring of turbulent bioconvection.

Under adjusted experimental conditions, open-to-air cultures of lux gene-engineered Ralstonia eutropha (wholecell biosensors of copper) exhibit bioconvection, which accounts for fluctuating bioluminescence. The power spectrum of bioluminescence intensity fluctuations recorded from a cylindrical sample 9 mm in diameter and approximately 10 mm in height is characterized by a dominant low-frequency oscillation (with a characteristic period of approximately 8-12 min), which is occasionally accompanied by a few weaker oscillations. The corresponding spectral peaks emerge on a high-noise background. The spectra of bioluminescence intensity fluctuations qualitatively resemble the spectra of temperature or fluid velocity fluctuations in an appropriate turbulent thermal convection system. It has been suggested that in a bioconvective system, like in thermal convection systems, the emergence of oscillation reflects the large-scale convective circulation that spans the height of the cylindrical cell. The velocity of large-scale bioconvective circulation was estimated to be 37-48 microm/s. The occasional emergence of weaker-than-dominant oscillations was explained through the coexistence and interaction of the large-scale circulation with, presumably, a gene-expression-related cyclic process (with a characteristic period of approximately 25-50 min).     

Synchronized oscillations in cytoplasmic free calcium concentration in confluent bradykinin-stimulated bovine pulmonary artery endothelial cell monolayers

Bradykinin-evoked rises in [Ca2+]i were measured in fura-2-loaded bovine pulmonary artery endothelial cell monolayers by dual wavelength excitation fluorimetry. In monolayers seeded thinly and grown to confluence, bradykinin, in the presence of external Ca2+, evoked a rise in [Ca2+]i composed of an initial peak and subsequent oscillating plateau. In the absence of external Ca2+, bradykinin evoked a rise in [Ca2+]i which then returned to the basal value without oscillating. In monolayers seeded near confluent density, the bradykinin-evoked peak in [Ca2+]i was followed by a steady plateau which showed no oscillation. The addition of the phorbol ester, phorbol 12,13-dibutyrate, to a monolayer during bradykinin-evoked oscillations abolished the oscillations and lowered [Ca2+]i partway back toward the basal level. The addition of the protein kinase C inhibitor, H7, did not abolish oscillatory activity, although the frequency of oscillation was reduced. These results indicate that synchronized oscillatory activity can occur in endothelial cell monolayers. It is suggested that these oscillations are dependent on intercellular coupling developed when the cells are grown to confluence and that the mechanism responsible for generating oscillations in [Ca2+]i requires extracellular Ca2+ and involves protein kinase C.