Metabolic Engineering 2, 149154 (2000)
doi:10.1006mben.1999.0141, available online at http:www.idealibrary.com on
NOTE
Effect of Glucose Analog Supplementation on Metabolic Flux
Distribution in Anaerobic Chemostat Cultures
of Escherichia coli
Susana J. Berr@ os-Rivera and Yea-Ting Yang
Department of Bioengineering and Chemical Engineering, Institute of Biosciences and Bioengineering,
Rice University, 6100 Main Street, Houston, Texas 77005-1892
George N. Bennett
Department of Biochemistry and Cell Biology, Institute of Biosciences and Bioengineering,
Rice University, 6100 Main Street, Houston, Texas 77005-1892
and
Ka-Yiu San 1
Department of Bioengineering and Chemical Engineering, Institute of Biosciences and Bioengineering,
Rice University, 6100 Main Street, Houston, Texas 77005-1892
Received June 17, 1999; accepted November 8, 1999
INTRODUCTION
Previous work in our laboratories investigated the use of methyl
:-glucoside (:-MG), a glucose analog that shares a phosphotransferase system with glucose, to modulate glucose uptake and therefore reduce acetate accumulation. The results of that study showed
a significant improvement in batch culture performance and a reduction in acetate excretion without any significant effect on the growth
rate in complex medium. The current study investigates the effect of
supplementing the culture medium with the glucose analog :-MG on
the metabolic fluxes of Escherichia coli under anaerobic chemostat
conditions at two different dilution rates. Anaerobic chemostat
studies utilizing complex media supplemented with glucose or glucose and :-MG at dilution rates of 0.1 and 0.4 h &1, were performed,
and the metabolic fluxes were analyzed. It was found that the
addition of the glucose analog :-MG has an effect on the specific
production rate of various extracellular metabolites. This effect is
slightly greater at the higher dilution rate of 0.4 h &1. However, the
glucose analog does not cause any major shift in the central
metabolic patterns. It was further observed that :-MG supplementation does not result in the reduction in specific acetate synthesis rate
in anaerobic chemostat cultures. These results emphasize the importance of testing different strategies for metabolic manipulation
under the actual operating conditions. 2000 Academic Press
1
To whom correspondence should be addressed. E-mail: ksan
rice.edu.
149
Escherichia coli is among the most widely used hosts for
the production of recombinant proteins. The goal of achieving high volumetric recombinant protein productivity
requires both high gene expression and high cell density.
However, these two requirements are difficult to meet
simultaneously because of the accumulation of harmful
acidic waste products such as acetate in dense cell cultures.
The accumulation of acetate above 1525 mM (Bauer et al.,
1990; Jensen and Carlsen, 1990; George et al., 1992) inhibits
cell growth and protein production. It is believed that the
cell balances any excess of carbon flux into its central
metabolic pathway that exceeds its needs for cell growth by
excretion of acidic metabolites with acetate as a major
component (Homs, 1986; El-Mansi and Homs, 1989).
Previous work in our laboratories explored the effect of
modulated glucose uptake on high-level recombinant
protein production as a means of reducing acetate
accumulation by limiting the carbon flux into the central
metabolic pathway of E. coli (Chou et al., 1994a, b). Two
different strategies were investigated. The first one involved
the use of a glucose analog, methyl :-glucoside (:-MG),
which shares a phosphotransferase system (PTS) with
glucose. This analog was used as a nontoxic competitive
1096-717600 35.00
Copyright 2000 by Academic Press
All rights of reproduction in any form reserved.
Note
Metabolic Engineering 2, 149154 (2000)
doi:10.1006mben.1999.0141
inhibitor to modulate cellular glucose uptake. The second
strategy involved the use of genetic engineering techniques
to modify cellular glucose uptake by constructing a mutation in ptsG, a gene encoding enzyme II in the glucose PTS
(Chou et al., 1994b). The results of these studies support
earlier observations that: (i) cells grown in complex medium
derive most building blocks from enriched sources, such as
yeast extract and casamino acids, and thus glucose is used
mainly for energy supply (Ingraham et al., 1983); (ii)
glucose uptake by E. coli is loosely regulated (Holms, 1986);
and (iii) uptake of glucose, when present in excess amounts,
normally exceeds the need for proper cell functions and subsequently leads to waste product formation, particularly
acetic acid (El-mansi and Holms, 1989). A significant
improvement in batch culture performance was observed as
a result of a reduction in acetate excretion without any
significant effect on the growth rate in complex medium.
The purpose of the current study was to examine the
effect of the addition of the glucose analog :-MG on the
metabolic fluxes of E. coli under anaerobic chemostat conditions at two different dilution rates or growth rates.
Anaerobic chemostat studies utilizing complex media
supplemented with glucose or glucose and :-MG were performed, and the metabolic flux distribution was analyzed.
Chemostat cultivation was chosen because it eliminates
any potential growth rate effect due to :-MG addition by
performing both experiments, with and without analog supplementation, at the same dilution rate. Furthermore, the
effect of growth rate was examined by performing the
chemostat experiments at two different dilution rates, 0.1 and
0.4 h &1. Chemostat cultivation also allows straightforward
evaluation of metabolic fluxes from experimental data. In
addition, our past experiences show that far more reliable flux
calculations can be obtained from chemostat data than from
batch cultures. Anaerobic conditions were chosen to study the
effect of the glucose analog supplementation because they
represent the worst case scenario of acetate accumulation.
MATERIALS AND METHODS
Bacterial Strain
The strain used in this study is GJT001, a spontaneous
S-(2-aminoethyl)-l-cysteine-resistant mutant of MC4100,
a 2lac strain (arg-lac) U169 rspL150 relA1 (Tolentino et al.,
1992). This strain was chosen because it is routinely used in
our laboratories.
Medium
LuriaBertani broth (LB) medium supplemented with
20 gL of glucose was used for the control runs, and LB
medium supplemented with 20 gL of glucose and 10 gL of
:-MG was used for the experimental runs. To reduce the
150
initial lag time that occurs under anaerobic conditions,
1 gL NaHCO3 was included in all runs. All media were
supplemented with 50 mgL streptomycin and 30 +LL
antifoam 289 (Sigma).
Bioreactor Conditions
The fermentations were carried under anaerobic
chemostat conditions at dilution rates of 0.1 and 0.4 h &1 in
a 2.5-L bioreactor (New Brunswick Scientific, Bioflo III)
initially with 1.3 L of medium during the anaerobic batch
stage and were maintained at 1.241.27 L working volume
for the anaerobic chemostat stage. The pH, temperature,
and agitation were maintained at 7.0, 32%C, and 250 rpm,
respectively. A constant flow of nitrogen (1215 mlmin)
was maintained through the fermentor headspace to establish anaerobic conditions. The continuous culture reached
steady-state after 4 to 6 residence times. Samples were taken
during the steady state phase at intervals of 5 h for the
0.1 h &1 dilution rate and at intervals of 2.5 h for the 0.4 h &1
dilution rate.
Analytical Techniques
Cell density (OD) was monitored at 600 nm in a spectrophotometer and reported as cell dry weight. Cell dry
weight was determined by collection of 100 or 50 ml of
culture in an ice bath. The samples were centrifuged at
4000g and 4%C for 10 min, washed with 0.15 M sodium
chloride solution, and dried in an oven at 55%C until they
reached a constant weight.
Fermentation broth samples were collected and centrifuged as described above. The supernatant was filtered
through a 0.45-+ m syringe filter and stored chilled for
further analysis. Acetate and ethanol were quantified using
a Varian 3000 gas chromatograph equipped with a porous
polystyrene column (Poropak QS, Alltech) and a flame
ionization detector with nitrogen as the carrier gas at
30 mlmin. The flame was maintained by a flow of hydrogen
and air at 30 and 300 mlmin, respectively. The injector and
detector temperatures were 215 and 245%C, respectively.
The column had a temperature profile to resolve the peaks.
Its initial temperature was 115%C for 3 min followed by a
3.5%Cmin ramp to 170%C. The column was held at 170%C
for 5 min followed by a 50%Cmin ramp to 240%C, and held
at 240%C for 10 min. The glucose analog :-MG, and
extracellular metabolites, such as succinate, lactate, formate, acetate, and ethanol, were quantified using an HPLC
system (Waters) equipped with a cation-exchange column
(HPX-87H, Bio-Rad Labs) and a differential refractive
index detector. A mobile phase of 2.5 mM H 2SO 4 solution
at a 0.6 mlmin flow rate was used and the column was
operated at 55%C.
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Metabolic Engineering 2, 149154 (2000)
doi:10.1006mben.1999.0141
Off-gases, specifically CO 2 and H 2, were analyzed offline
using the Varian 3000 gas chromatograph equipped with a
divinylbenzene porous polymer column (HayeSep DB,
HayeSep) and a thermal conductivity detector. The column,
injector, detector, and filament temperatures were 28, 28,
140, and 225%C, respectively. Helium was used as the carrier
gas at a flow rate of 30 mlmin. The glucose and extracellular pyruvate concentrations were assayed enzymatically
using the hexokinase kit and pyruvate kit from Sigma. For
the determination of intracellular pyruvate, samples were
taken from the reactor sample port and processed
immediately. Ten milliliters of the collected sample was centrifuged as described above. The cells were resuspended in
minimal medium supplemented with 20 mM glucose and
centrifuged. The glucose is used as a tracer to calculate
interstitial volume (Grosz and Stephanopoulos, 1990; Yang
et al., 1999). Six percent perchloric acid (PA) was added
with a ratio of [OD 600 nm_V sample(ml)]V PA(ml)=40 to
FIG. 1. Summary of anaerobic chemostat experimental results for LB supplemented with 20 gL glucose or 20 gL glucose and 10 gL :-MG at
constant dilution rates of 0.1 and 0.4 h &1.
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lyse cells and inactivate enzymes. The samples with PA were
kept on ice for 20 min, neutralized with 3 M KHCO 3 at a
KHCO 3: PA volume ratio of 0.3:1, and centrifuged for 10
min. Supernatants were assayed enzymatically for pyruvate
determination using the Sigma pyruvate kit. To compare
the intracellular pyruvate concentrations, the cytoplasmic
volume was assumed to be constant at 1.6 + lmg dry.
Previous reported values for cytoplasmic volume varied
from 1.0 to 2.1 +lmg dry depending on the strain tested
(Booth et al., 1979; Stock et al., 1977).
FIG. 2.
RESULTS AND DISCUSSION
Figure 1 shows the raw data of the anaerobic chemostat
runs at 0.1 and 0.4 h &1 dilution rates, including cell density,
amount of glucose consumed, intracellular pyruvate level,
and concentrations of different fermentation products.
From these results, it is interesting to note that at the
0.1 h &1 dilution rate, the addition of :-MG caused a
decrease of only 110 in cell density, while at the 0.4 h &1
dilution rate, the cell density decreased by 520. This was
Central anaerobic metabolic pathway of E. coli.
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followed by a similar trend in glucose consumption.
Glucose uptake decreased by 180 with the addition of
:-MG at 0.1 h &1 dilution rate and by 610 at 0.4 h &1. These
observations suggest that the glucose analog :-MG, has a
greater impact on glucose uptake at higher dilution rates.
On the other hand, the intracellular pyruvate concentration
dropped with the addition of the glucose analog at both
dilution rates, but more drastically at 0.1 h &1 (710) than at
0.4 h &1 (310); this trend is opposite to that of glucose
uptake. At the 0.1 h &1 dilution rate, the concentrations of
acetate, ethanol, formate, and lactate decreased by 4, 19, 27,
and 180, respectively, while that of succinate increased by
70. At the 0.4 h &1 dilution rate, the concentrations of
acetate, ethanol, and formate decreased by 46, 43, and 530,
respectively, while that of succinate remained relatively
unchanged. For both experiments, the lactate levels were
undetectable at the 0.4 h &1 dilution rate. The larger
decrease in the extracellular metabolite concentrations at
the 0.4 h &1 than at the 0.1 h &1 dilution rate with the addition of :-MG might be due to the more drastic decrease in
cell density at the higher dilution rate. The intracellular
pyruvate concentration decreases with dilution rate (Fig. 1).
In addition, the intracelluar pyruvate concentration also
decreases in the presence of the glucose analog for the same
dilution. This is probably a result of the observed decrease
in glucose uptake.
The central anaerobic metabolic pathway of E. coli is
depicted in Fig. 2. The specific rates, the fluxes in mmol
g-cell h, of some of the major reaction pathways are
calculated. The results of four different runs at two different
dilution rates are shown in Table 1.
TABLE 1
Effect of :-MG Supplementation on Metabolic Flux Distribution
for Anearobic Chemostat Cultivation in Complex Media at Dilution
Rates of 0.1 and 0.4 h &1
Glucose uptake (& 1 )
Pyruvate formation (& 5$ )
Succinate formation (& 6 )
Lactate formation (& 7 )
Formate excretion (&form )
Ethanol formation (& 10 )
Acetate formation (& 11 )
Acetyl-CoA formation (& 8 )
D=0.1 h &1
D=0.4 h &1
Glucose
Glucose +:-MG
Glucose
Glucose +:-MG
3.91
7.89
0.70
0.21
2.16
3.92
3.76
7.68
3.58
7.78
0.84
0.19
1.77
3.55
4.04
7.59
26.00
19.90
1.49
nd
8.57
10.50
9.41
19.9
Metabolic Flux Analysis: Effect of :-MG at 0.1 h &1 Dilution
Rate
At a dilution rate of 0.1 h &1, the results do not show any
major change in the metabolic patterns with the addition of
the glucose analog, even though there is a slight decrease of
80 in the specific glucose uptake rate. No acetate reduction
was observed in the presence of the glucose analog; instead
a slight increase of 70 was found and was accompanied by
a decrease of 90 in the ethanol flux. In general, the succinate flux increased by 200, while all other fluxes increased
or decreased by less than 100. These results suggest that the
addition of :-MG to anaerobic chemostat cultures at this
low dilution rate has only a minor effect on the metabolic
flux distribution of E. coli.
Metabolic Flux Analysis: Effect of :-MG at 0.4 h &1 Dilution
Rate
The addition of :-MG has a slightly greater effect on
the metabolic patterns at the 0.4 h &1 dilution rate; most
of the changes in metabolic fluxes observed were greater
than 100. The glucose flux decreased by 200, while the
pyruvate flux increased by 120 and the succinate flux
doubled. The steeper decrease in the glucose flux compared
to that at the 0.1h &1 dilution rate is probably due to the
observed increase in :-MG uptake (data not shown). The
increase in the specific :-MG uptake rate is also likely the
cause for the relatively sharp drop in the observed biomass
concentration (Fig. 1) as more energy is being spent on
transporting the inert analog. At the pyruvate branch point,
the lactate flux was not observed and the acetyl-CoA flux
increased by 120. At the acetyl-CoA node, the ethanol
specific flux increased by 140 while the acetate flux
increased by 90. At this higher dilution rate, the effect of
the glucose analog on the metabolic flux distribution of E.
coli is larger than that at the 0.1 h &1 dilution rate, but still
there is no major shift in the basic metabolic patterns.
CONCLUSION
It was found that the addition of the glucose analog
:-MG had an effect on the specific production rate of
various extracellular metabolites. This effect was slightly
greater at the higher dilution rate of 0.4 h &1. However, the
glucose analog did not cause any major shift in the central
metabolic patterns. In both cases, the addition of the
glucose analog did not lead to the reduction in acetate
accumulation observed in previous studies (Chou et al.,
1994a) in batch cultures, in which the specific growth rate
varied from about 1.4 h &1 at the beginning of the fermentation to about 0 toward the end. On the contrary, a small
increase in acetate specific flux was observed. These results
20.59
22.27
2.97
nd
8.00
11.98
10.29
22.7
Note. Fluxes (mmolg-cell h) are calculated from data in Fig. 2, The
value & 5$ is estimated by & 5$ =& 7 +& 8 , and & 8 is estimated by & 8 =& 10 +& 11 ,
nd, not detected.
153
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Metabolic Engineering 2, 149154 (2000)
doi:10.1006mben.1999.0141
emphasize the importance of testing the different strategies
for metabolic manipulation under the actual operating
conditions. In batch cultures, the cells are exposed to timevarying conditions, with maximal glucose exposure at the
beginning. However, in chemostat cultures, the cells are
exposed to a constant glucose concentration. In addition,
the acetate concentration commonly reported in batch
cultures is actually the integration of acetate production
rate over the entire fermentation period. Note that the
acetate production rate in the presence of :-MG at D=
0.4 hr &1 was greatly reduced (11.5 vs 6.3 mmolL-h) due to
a significant decrease in the biomass. It is possible that the
observed lower acetate accumulation (Chou et al., 1994) is
due to a reduced acetate production rate and not to a
specific production rate.
It can be concluded from the results of this study that
although the use of a glucose analog to modulate glucose
uptake might be a good strategy for reducing acetate accumulation in batch cultures, it will not offer any significant
advantage under chemostat conditions. These differences
may be explained by the difference in the growth environment and conditions the cells are exposed to in a batch
versus a chemostat cultivation. Whereas in a chemostat
bioreactor the specific growth rate equals the dilution rate
and is fixed externally, in a batch culture it is dependent
on the strain and medium composition. In addition, the
transient nature of the batch cultivation implies that the
concentrations of both substrates and metabolites vary constantly with time, while at steady state these concentrations
are time-invariant for a chemostat culture. Specifically, the
cells are exposed to a very rich environment for most of the
time during batch cultivation, whereas they are always
under limiting environment under a chemostat setting.
ACKNOWLEDGMENTS
This study was supported in part by National Science Foundation
Grants BES-9305797 and BES-9411928. This material was also based
on work supported under a National Science Foundation Graduate
Fellowship.
154
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