Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate

Acetate accumulation under aerobic conditions is a common problem in Escherichia coli cultures, as it causes a reduction in both growth rate and recombinant protein productivity. In this study, the effect of replacing the glucose phosphotransferase transport system (PTS) with an alternate glucose transport activity on growth kinetics, acetate accumulation and production of two model recombinant proteins, was determined. Strain VH32 is a W3110 derivative with an inactive PTS. The promoter region of the chromosomal galactose permease gene galP of VH32 was replaced by the strong trc promoter. The resulting strain, VH32GalP+ acquired the capacity to utilize glucose as a carbon source. Strains W3110 and VH32GalP+ were transformed for the production of recombinant TrpLE-proinsulin accumulated as inclusion bodies (W3110-PI and VH32GalP+-PI) and for production of soluble intracellular green fluorescent protein (W3110-pV21 and VH32GalP+-pV21). W3110-pV21 and VH32GalP+-pV21 were grown in batch cultures. Maximum recombinant protein concentration, as determined from fluorescence, was almost four-fold higher in VH32GalP+-pV21, relative to W3110-pV21. Maximum acetate concentration reached 2.8 g/L for W3110-pV21 cultures, whereas a maximum of 0.39 g/L accumulated in VH32GalP+-pV21. W3110-PI and VH32GalP+-PI were grown in batch and fed-batch cultures. Compared to W3110-PI, the engineered strain maintained similar production and growth rate capabilities while reducing acetate accumulation. Specific glucose consumption rate was lower and product yield on glucose was higher in VH32GalP+-PI fed-batch cultures. Altogether, strains with the engineered glucose uptake system showed improved process performance parameters for recombinant protein production over the wild-type strain.     

Enhanced production of plasmid DNA by engineered Escherichia coli strains

Escherichia coli strains VH33 (PTS? GalP? strain displaying a strongly reduced overflow metabolism) and VH34 (additionally lacking the pyruvate kinase A) were evaluated for the production of a plasmid DNA (pDNA) vaccine. The parent (W3110) and mutant strains were cultured using 10 g of glucose/L. While the specific growth rates of the three strains were similar, they presented differences in the accumulation of acetate. W3110 accumulated up to 4 g/L of acetate, VH33 produced 1.4 g/L, and VH34 only 0.78 g/L. VH33 and VH34 produced 76% and 300% more pDNA than W3110. Moreover, VH34 demanded 33% less oxygen than VH33 and W3110, which can be advantageous for large-scale applications.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.     

Vitreoscilla hemoglobin expression in engineered Escherichia coli: improved performance in high cell-density batch cultivations

High cell-density cultivations are the preferred system for biomolecules production by Escherichia coli. It has been previously demonstrated that a strain of E. coli with a modified substrate transport system is able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. The use of elevated amounts of glucose from the beginning of the cultivation, eliminates the existence of substrate gradients due to deficient mixing at large-scale. However, the large amounts of oxygen demanded resulted in microaerobic conditions after some hours of cultivation, even at small-scale. In this work, the effect of expressing the Vitreoscilla hemoglobin (VHb) in the engineered strain during batch cultures using high-glucose concentrations was tested. Together, the expression of VHb and the modified substrate transport system resulted in a 33% increase of biomass production compared to the parental strain (W3110) lacking the VHb in batch cultivations using 25 g/L of glucose. When 50 g/L of glucose were used, expression of VHb in the modified strain led to 11% higher biomass production compared to W3110. The VHb also increased the growth rates of the strains by about 30% in the aerobic phase and more than 200% in the microaerobic phase of batch cultivation.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.     

Enhanced beta-galactosidase production by high cell-density culture of recombinant Bacillus subtilis with glucose concentration control

Cell concentration, recombinant protein (beta-galactosidase) level, and the specific enzyme expression level were increased from 19 to 184 g/L, 18.3 to 129 U/mL, and 3.2 to 5.7 U/mg protein, respectively, in fed-batch culture of recombinant Bacillus subtilis when glucose concentration was controlled at 1 g/L as compared with those of conventional fed-batch culture. Glucose concentration of the culture broth was monitored by an automatic on-line glucose analyzer and controlled with a moving identification combined with optimal control (MICOC) strategy. When glucose concentrations were controlled at 10, 1, and 0.2 g/L, accumulated propionic acid concentrations and specific enzyme activities were 18.5, 4.4, and 0.6 g/L and 2.9, 5.7, and 7.1 U/mg protein, respectively. The addition of various concentrations of sodium propionate to the growth medium in batch cultures resulted in a drastic decrease in the growth rate with respect to propionate concentration. The propionic acid was shown to be responsible for cell growth inhibition and enzyme activity reduction in fed-batch culture. (c) 1992 John Wiley & Sons, Inc.     

Switching the mode of sucrose utilization by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H⁺ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.     

Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites.     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture

The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea. In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source. As a result, a maximum cell dry weight concentration of 48 g/L was achieved. This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions. In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed-batch mode to establish a nitrogen-limited culture. Such a modification resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the N-sufficient culture. The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This N-limited fed-batch culture was successfully scaled up from 3.7 L to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L). The maintenance of the culture at a higher temperature (i.e., 32 degrees C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed-batch culture (30 L) without such a treatment. The enhancement of lutein production resulted from the combination of nitrogen limitation and high-temperature stress.     

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.     

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.     

Changes in intracellular metabolite pools, and acetate formation in Escherichia coli are associated with a cell-density-dependent metabolic switch

A cell density-dependent metabolic switch in amino acid metabolism occurs in E. coli W3110 batch cultures at 1.15 g dry wt l^–1 (Han L, Doverskog M, Enfors S-O, Häggström L, 2002, J. Biotechnol. 92: 237–249). A two- to three-fold decrease of the concentration of most glycolytic and citric acid cycle metabolites, and an increase in acetyl-CoA concentration after the switch, indicates that the central metabolism also is affected. The specific acetate production rate decreases throughout the culture, except for a temporary increase at the switch point. The intracellular acetate concentration remains relatively constant during the culture.     

Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products

In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.