Metabolic Flux Analysis of Escherichia
coli Expressing the Bacillus subtilis
Acetolactate Synthase in Batch and
Continuous Cultures
Aristos A. Aristidou,1,* Ka-Yiu San,1 George N. Bennett2
1
Department
2
of Bioengineering and Chemical Engineering and
Department of Biochemistry and Cell Biology, Institute of Biosciences and
Bioengineering, Rice University, P.O. Box 1892, Houston, Texas 77251-1892
Received 23 July 1998; accepted 30 November 1998
Abstract: Metabolically engineered Escherichia coli expressing the B. subtilis acetolactate synthase has shown
to be capable of reducing acetate accumulation. This reduction subsequently led to a significant enhancement in
recombinant protein production. The main focus of this
study is to systematically examine the effect of ALS in
the metabolic patterns of E. coli in batch and continuous
culture. The specific acetate production rate of a strain
carrying the B. subtilis als gene is 75% lower than that of
the control strain (host carrying the control plasmid pACYC184) in batch cultures. The ALS strain is further demonstrated to be capable of maintaining a reduced specific
acetate production rate in continuous cultures at dilution
rates ranging from 0.1 to 0.4 h−1. In addition, this ALS
strain is shown to have a higher ATP yield and lower
maintenance coefficient. The metabolic flux analysis of
carbon flux distribution of the central metabolic pathways and at the pyruvate branch point reveals that this
strain has the ability to channel excess pyruvate to the
much less toxic compound, acetoin. © 1999 John Wiley &
Sons, Inc. Biotechnol Bioeng 63: 737–749, 1999.
Keywords: Escherichia coli; metabolic flux analysis; ALS
INTRODUCTION
Escherichia coli is extensively used in industry as a host for
recombinant protein production. The ease of genetic manipulation and wealth of available genetic information
coupled with fast growth rate, standardized cultivation techniques and cheap media are reasons for its popularity. In the
high density cultures favored by industry can lead to accumulation of acetate and cessation of growth.
The reduction of acetate production is of primary concern
in fermentation and recombinant protein production by E.
coli. Acetate is produced in glucose fermentation by aerobic
acetogenesis or through the Crabtree effect (Doelle, 1975).
Acetate formation is due to excess influx of carbon from
glucose that the cell is unable to utilize for biomass synthe-
sis, leading to repression of the TCA cycle enzymes by
glucose, or uncoupled metabolism (Holms, 1986; Majewski
and Domach, 1990). Anaerobically, acetate production provides an additional mole of ATP per mole of glucose. Production of acetate represents a loss from carbon flux to cell
growth as well as recombinant protein production. Acetate
can also act as a liphophilic agent which can dissipate the
pH component of proton motive force (PMF). Several previous reports have shown that short-chain fatty acids repress
synthesis of DNA, RNA, protein, lipids, and peptidoglycans
(Cherrington et al., 1990, 1991). These components of the
cellular machinery are necessary for growth as well as protein production. Not surprisingly, the reduction of acetate
accumulation has also been shown to enhance recombinant
protein production (Jensen and Carlsen, 1990).
Previously, our laboratories have reported that significant
reduction in acetate accumulation can be achieved by expressing biologically active B. subtilis acetolactate synthase
(ALS) in E. coli. Such metabolically engineered E. coli are
able to channel excess carbon to acetoin; and this byproduct
was further shown to be much less toxic than acetate (Aristidou et al., 1994). In addition, the ALS strain has been
demonstrated to be capable of enhancing recombinant protein productivity in E. coli (Aristidou et al., 1995).
In this work, we focus on a study of the effects of B.
subtilis ALS on the central metabolic fluxes in E. coli using
both batch and continuous cultures. In addition, the effect of
growth rate on the redistribution of metabolic flux was systematically examined at various dilution rates ranging from
0.1 to 0.4 h−1.
MATERIALS AND METHODS
Bacterial Strain and Plasmids
Correspondence to: K.-Y. San
* Present address: VTT Biotechnology and Food Research, Tietotie 2,
PL 1501, 02044 VTT, Finland
Contract grant sponsor: National Science Foundation
Contract grant numbers: BCS 9315797; BES 9411928
© 1999 John Wiley & Sons, Inc.
E. coli GJT001 was used throughout this study. The ALS
strain carried plasmid pAAA215 that expresses the B. subtilis acetolactate synthase (ALS) (Aristidou et al., 1994). It
contains the relaxed replicon p15A and the tetracycline re-
CCC 0006-3592/99/060737-13
sistance gene derived from pACYC184 (Chang and Cohen,
1978). GJT001 carrying the vector pACYC184 was used as
a control.
Cultivation System
All cultivations took place in Luria–Bertani broth (LB)
supplemented with 20 g L−1 of glucose. For batch experiments, including the start-up media for the chemostat, 1 g
L−1 of NaHCO3 was also added to reduce the initial lag
period characteristic of anaerobic growth. Tetracycline and
streptomycin were added at concentrations of 12.5 and 30
mg L−1, respectively in all media. Fermentations were conducted in a 2.5-L benchtop fermentor (New Brunswick Scientific, BioFlo III), with a working volume of 1.5 and 1.25
L for batch and chemostat cultivations, respectively. The
pH, temperature, and agitation speed were maintained at
7.0, 32 °C, 225 rpm, respectively. Anaerobic conditions
were established by gassing the growth media with N2 prior
to inoculation, and after that maintained by a constant N2
flow through the fermentor headspace. For continuous culture steady state is established after the elapse of 5–6 residence times. Experimental data corresponding to a particular dilution rate is the average of three samples.
polymer column (HayeSep DB, HayeSep) and a thermal
conductivity detector. The column and injector were operated at 28°C with helium as the carrier gas at a flowrate of
30 mL min−1. The detector and filament temperatures were
set at 140 and 225°C, respectively.
Methodology of Flux Determination
Metabolic Network
The metabolic network that describes the central carbon
fluxes in anaerobically grown E. coli is shown schematically in Fig. 1 with the relevant enzymatic reactions listed in
Appendix A. Table I summarizes the stoichiometry of the
various enzymatic reactions. Pyruvate is an important
branchpoint in this network, where carbon flows to either
acetyl-CoA with the concomitant formation of formic acid,
lactate, or to acetolactate for cells expressing the ALS enzyme. Acetyl-CoA is subsequently channeled to the synthesis of acetate or ethanol.
The fate of formate will depend on the prevailing medium
pH among other factors. As shown in Table II, the break-
Analytical Techniques
Cell density was routinely monitored spectrophotometrically at 600 nm. In addition, cell dry weight was periodically determined from a 100-mL sample volume. For this
analysis, the culture was first cooled in ice bath, washed,
centrifuged at 8000g and 4°C for 10 min and dried in an
oven for about 8 h. For metabolite analysis, fermentation
samples were collected as above, and the supernatant was
subsequently filtered through a 0.45-micron syringe filter
and stored at −20°C until analyzed.
Glucose was assayed enzymatically (Sigma Hexokinase
kit). Acetate, ethanol, and acetoin were quantified using a
Varian 3000 gas chromatograph equipped with a porous
polystyrene column (Poropak QS, Alltech) and a flame ionization detector (FID). Nitrogen was the carrier gas at 30
mL min−1. The flame was maintained by H2 and air at 30
and 300 mL min−1 respectively. The injector and detector
temperatures were at 215 and 245°C, respectively. The following temperature profile was used: initially at 115°C for
3 min followed by a 3.5°C min−1 ramp to 170°C and held at
170°C for 10 min. HPLC analysis was also employed in
order to confirm enzymatic and GC analyses, as well as to
detect any additional metabolites present in the fermentation
broth. The HPLC system was equipped with a cationexchange column (HPX-87H, Bio-Rad) and a differential
refractive index detector (Waters). A mobile phase of 2.5
mM H2SO4 solution at a 0.6 mL min−1 flow rate was used,
and the column was operated at 55°C.
Off-gas analysis (CO2 and H2) was conducted by a gas
chromatograph equipped with a divinylbenzene porous
738
Figure 1. Central metabolic pathways of anaerobically grown E. coli.
The seven principal fermentation end products are represented in boldface,
along with acetoin produced by cells expressing the ALS enzyme. Circled
numbers correspond to the enzymatic reactions in Appendix A.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 63, NO. 6, JUNE 20, 1999
Table I.
Stoichiometric flux relationships for anaerobic growth.a
i
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Metabolite
Acetate
Acetoin
Ethanol
Formate
Glucose
Lactate
Succinate
CO2
H2
C
atoms
O/R
2
4
2
1
6
4
4
1
0
0
−2
−2
+1
0
0
+1
+2
−1
Net conversion: ri (t)
v11
v12
v10
v8 − v9
v1 ⳱ (CF, gluc − Cglc) × (D/X)
v7
v6
−v6 + v9 + 2v12 ⳱ CER
v9
Acetyl-CoA
Glucose 6-P
Glyceraldehyde 3-P
PEPb
Pyruvate
v8 − v10 − v11
v1 − v2 − v3
2v3 − v4
−(v1 + v5) + v4 − v6 ⳱ v5⬘ + v4 − v6
v5 − v7 − v8 − 2v12
NADH (rNADH)
ATP (rATP,␹)c
v4 − 2v6 − v7 − 2v10
⳱ 3v3 + v11 − vm ⳱ rATP − me
O/R balance
C to fermented byproducts
C to biomass d
−v6 + v8 − 2v10 + 2v12
3v6 + 4v7 + v8 + 2v10 + 2v11 + 6v12
v2
a
Species 1–9 correspond to measurable quantities, and species 10–14 represent metabolic intermediates.
b
v5⬘ ⳱ v1 + v5 ⳱ overall flux of PEP to pyruvate.
c
rATP, ␹ ⳱ ATP synthesis adjusted for maintenance ⳱ rATP − me.
d
Assume that glucose not fermented to extracellular products is converted to biomass.
down of formate to H2 and CO2 is minimal (0.3%) at pH
7.8, in contrast to pH 6.2 where most of this compound
(87%) is converted to H2 and CO2 (Blackwood et al., 1956).
Furthermore, the induction of formate hydrogen-lyase has
been correlated to formate accumulation (Pecher et al.,
1983). A biphasic formate profile, consisting of an initial
accumulation phase followed by degradation, has been reported for anaerobic batch growth of E. coli in complex
glucose media (Diaz-Ricci et al., 1990). Hence, the rate for
formate breakdown (␯9) cannot be assumed and has to be
estimated from experimental data by including this reaction
in the metabolic network.
Flux Determination
A metabolic matrix is constructed based on the law of mass
conservation and on the pseudo-steady state hypothesis
(PSSH) on the intracellular intermediate metabolites. The
formulation resulted in a set of linear equations that can be
Table II. Effect of pH on the conversion of formate to CO2 and H2.a
pH
Formate
Acetate
Ethanol
H2 (v9)
v9 /v8 (%)
CO2
6.2
7.8
2.43
86.0
36.5
38.7
49.8
50.5
75.0
0.26
87.0
0.3
88.0
1.75
a
Values are in mmoles of product formed per 100 mmol glucose fermented (Blackwood 1956). Flux (v8) corresponds to the total flux through
PFL ≈ acetate + ethanol.
expressed as a stoichiometric matrix A of dimension m by n
with vectors for net accumulation, r (m × 1) and metabolic
flux, ␯ (n × 1). Theoretically, two different matrices can be
formed using slightly different assumptions for the twelve
metabolic fluxes (␯1 to ␯12 for this case). The first one based
solely on the measured extracellular metabolites and PSSH
balances on the intracellular intermediate metabolites resulting in a square matrix (n ⳱ m ⳱ 12). The additional CO2
and H2 measurements produce an overdetermined system
by which the flux calculation may be verified (m ⳱ 14). In
the latter case the solution can be found by a least-squares
fit. A more detailed discussion on flux determination as well
as CO2 and H2 measurements in a chemostat culture can be
found elsewhere (Aristidou, 1994).
RESULTS AND DISCUSSION
Anaerobic Batch Cultures
The metabolic fluxes were determined for batch anaerobic
cultures. Time profiles from these experiments are shown in
Fig. 2, where growth characteristics of cells harboring the
als gene are compared with those of the host itself and also
the host carrying the control plasmid pACYC184. The specific growth rates as well as final cell densities appear to be
comparable among the three systems. As expected for anaerobic growth, the cells are excreting large quantities of the
partially oxidized byproducts acetate, formate, and ethanol.
ARISTIDOU, SAN, AND BENNETT: METABOLIC FLUX ANALYSIS OF E. COLI EXPRESSING B. SUBTILIS ALS
739
Table III. Effect of ALS on the metabolic flux distribution.a
Plasmid
Flux
To
None
pACYC184
pAAA215
v1
v2
v3
v4
v5⬘
v6
v7
v8
v9
v10
v11
v12
rATP
rCO2
Glucose 6-P
Biosynthesis
Glyceraldehyde-3-P
PEP
Pyruvate
Succinate
Lactate
Formate
H2
Ethanol
Acetate
Acetoin
ATP
CO2
7.64
1.39
6.25
12.50
12.09
0.41
0.00
12.09
6.43
6.44
5.65
0.00
24.40
6.02
8.12
1.25
6.88
13.75
13.28
0.47
0.00
13.28
7.72
6.25
7.03
0.00
27.66
7.85
10.61
3.63
6.97
13.95
11.59
2.36
0.00
8.79
1.70
6.94
1.85
1.40
22.77
2.13
a
Fluxes (mmol (g-cell h)−1) calculated for cells grown anaerobically in
batch cultures.
Figure 2. Time profiles of cell growth, glucose consumption, and excretion of glycolytic byproducts. Batch anaerobic culture in LB + 2% glucose
at 30°C. Flux calculations were based on data taken between 4 and 9 h.
GJT001 (circle); GJT001:pACYC184 (open triangle); GJT001:pAAA215
(alsS) (filled triangle).
What sets apart the ALS strain is the diminished acetate
production profile. Acetoin formation commences at the
point where acetate levels reach about 10 mM, and from
then on the rate of acetate excretion declines substantially.
The final acetate level for the ALS strain approaches 20
mM, compared with 60–70 mM for the other two strains.
Formate accumulation profiles for the three strains are very
similar for the first 8 h. However, the final formate concentration for the ALS strain is about 30% lower.
The estimated fluxes (mmol (g-cell h)−1) for the batch
experiments are summarized Table III. These values represent the average of 4–5 sampling points about the midexponential growth phase and are within the balanced
growth period. Calculations that span the entire fermentation period indicate that indeed the time dependency of the
fluxes is smallest during this interval (data not shown). Major differences between the three systems again is in the
acetate flux, ␯11, which is 65% and 75% lower for the ALS
strain compared to the host and the host bearing the control
plasmid, respectively. Furthermore, the ALS strain appears
to have significantly reduced hydrogen and CO2 production
rates.
For all three systems (i.e. cells harboring the als gene, the
host itself and also the host carrying the control plasmid
740
pACYC184), glucose uptake rates, ␯1, are 3–5 times higher
when compared with similar aerobic batch cultures (data not
shown). This is to be expected since under anaerobic conditions the cell’s efficiency for energy production is diminished considerably: the ATP yield on glucose is 2.5–3.5 mol
ATP per mol glucose (␯ATP/␯1 vs 38 normally derived from
aerobic respiration). The ATP yields will be discussed further in a later section.
The high glycolytic fluxes result in a high generation of
NADH (␯4) that the cell needs to recycle for metabolism to
continue. In the absence of detectable lactate formation and
without a significant quantity of succinate formed, the ethanol pathway fulfills the requirement for NADH regeneration
in all strains. As shown in Table I, the redox balance is
accomplished by having ␯4 » 2␯10. Thus, despite the fact
that the ALS enzyme reduces the flux to acetyl-CoA (␯8),
ethanol production remains unaffected due probably to superimposed constraint of recycling reduced NADH. In other
words, the partition of acetyl-CoA into ethanol or acetate is
determined primarily by the cells oxidoreductive requirements. The acetyl-CoA branchpoint would be classified as a
“weakly rigid node” according to the terminology introduced by Stephanopoulos and Vallino (1991), characterized
by a combination of a dominant (ethanol) and a subordinate
(acetate) branch.
The increased flux through succinate by the ALS strain (2
mmol (g-cell h)−1) is probably a further indication of the
cell’s attempt to balance its redox status. The need of maintaining redox neutrality is an important consideration in
designing new pathways, especially when these pathways
are themselves coupled to NADH generation or consumption. For example, the changes in the fermentation characteristics of E. coli expressing the pet operon from Z. mobilis,
was attributed to the modification of the intracellular redox
state (Ingram and Conway, 1988).
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 63, NO. 6, JUNE 20, 1999
Anaerobic Chemostat Cultures
In batch cultivation cells are grown at their maximum rate in
an environment that changes progressively with time as a
result of metabolic activity. There are at least two distinct
transition zones in the time course of batch cultivation: one
zone from the zero to maximum growth rate and a retardation phase extending from the exponential to the stationary
phase. The changing growth environment, both in terms of
excreted byproducts as well as depletion of important nutrients, can lead to profound physiological changes with
time. Chemostats, on the other hand, can provide a defined
and steady state environment and thus more reliable and
reproducible data (Dawson, 1984).
The levels of various extracellular metabolites and biomass for the series of dilution rates is shown in Fig. 3, and
the corresponding calculated fluxes are summarized in
Table IV and in Fig. 4. For each strain seven dilution rates
ranging from 0.1 to 0.4 were performed, with the latter
approaching the washout dilution rate for the given growth
conditions. For aerobic E. coli cultures Meyer et al. (1984)
found the critical dilution rate for the onset of acetate formation to be 0.35 and 0.2 h−1 for defined and complex
media, respectively. As indicated in Fig. 3, overflow me-
Table IV.
Effect of ALS on metabolic flux distribution.a
D (h−1)
Flux
v1
v2
v3
v4
v5⬘
v6
v7
v8
v9
v10
v11
v12
rCO2
ALS
0.10
0.15
0.20
0.25
0.30
0.35
0.40
+
−
+
−
+
−
+
−
+
−
+
−
+
−
+
−
+
−
+
−
+
−
+
−
+
−
3.86
4.59
1.29
0.46
2.57
4.11
5.14
8.21
4.15
6.90
0.99
1.31
0.09
0.48
3.06
6.43
1.87
5.02
2.31
3.27
0.76
3.16
0.50
0.00
1.88
3.71
5.83
5.53
2.21
0.57
3.62
4.96
7.23
9.91
6.32
9.06
0.91
0.86
0.15
1.87
4.84
7.19
2.74
4.83
3.87
3.69
0.97
3.50
0.66
0.00
3.15
3.97
7.21
6.47
2.99
0.73
4.22
5.74
8.43
11.47
7.39
10.77
1.04
0.70
0.19
2.05
5.74
8.72
2.98
4.36
4.79
4.46
0.95
4.26
0.74
0.00
3.42
3.66
8.22
7.96
2.61
0.53
5.62
7.44
11.23
14.87
10.31
13.84
0.92
1.03
0.24
1.15
7.80
12.70
3.66
5.26
6.53
6.37
1.47
6.33
1.04
0.00
4.82
4.23
10.36
9.48
2.63
1.29
7.73
8.20
15.46
16.39
13.96
15.27
1.50
1.12
0.73
0.73
9.98
14.54
4.46
6.12
7.65
7.04
2.33
7.50
1.63
0.00
6.21
5.00
11.51
14.19
1.78
1.45
9.73
12.71
19.45
25.43
18.21
23.81
1.24
1.61
1.70
0.73
12.50
23.09
5.43
8.21
9.04
11.22
3.45
11.86
2.01
0.00
8.21
6.59
12.94
17.37
1.42
1.17
11.52
16.20
23.05
32.40
21.25
30.30
1.79
2.10
1.88
1.15
14.17
29.15
5.63
11.64
10.90
14.37
3.27
14.78
2.60
0.00
9.04
9.54
a
Anaerobic chemostat cultivation in rich glucose media. Fluxes expressed in (mmol (g-cell h)−1).
Figure 3. Summary of chemostat experimental results for dilution rates
0.1–0.4 h−1. Anaerobic growth in LB media supplemented with 2% glucose
at 32°C. GJT001:pACYC184 (control, open bars); GJT001:pAAA215
(ALS, filled bars).
tabolism is very prominent in anaerobically growth E. coli,
and it can provide additional energy (acetate) or a means for
disposing excess NADH (ethanol, succinate, lactate). At
dilution rates higher than 0.20 h−1, the biomass concentration starts to decline whiles residual glucose amounts begin
to rise with the dilution rate. However, cell densities for the
ALS strain are higher than the control for almost all dilution
rates. An important difference between the two strains is the
level of acetate that is maintained significantly lower for the
ALS strain compared with control. The steady state ethanol,
succinate, acetate and acetoin concentrations decrease with
increasing dilution rates for both strains. The lactate remains
rather constant for the ALS strain while exhibiting a maximum concentration at a dilution rate of approximately 0.15
h−1 for the control strain. The formate, on the other hand,
has a peak concentration at around D ⳱ 0.25 h−1 for both
strains.
The fluxes show an increasing trend with the dilution rate
(Fig. 4) and are qualitatively similar with the those from the
batch cultures. Quantitatively, chemostat fluxes at D ⳱ 0.3
h−1 approach those of the batch cultivation. It is interesting
to note that the specific growth rate during the sampling
period for the batch experiments was actually between 0.25
and 0.3 h−1.
The greatest impact of the ALS enzyme is the reduction
in acetate and lactate, and to a lesser extent formate accu-
ARISTIDOU, SAN, AND BENNETT: METABOLIC FLUX ANALYSIS OF E. COLI EXPRESSING B. SUBTILIS ALS
741
Figure 4. Summary of flux analysis results for dilution rates of 0.1, 0.2, 0.3, and 0.4 h−1 for the control and the ALS strains. Insert shows the fluxes of
ethanol (␯10) and acetate (␯11) for the control (−) and ALS (+) strains as functions of the dilution rates.
mulation. The carbon flux to acetate (␯11) is diminished by
70–80% for all dilution rates with a corresponding reduction
in the steady-state extracellular acetate concentration. The
ALS strain is capable of maintaining a reduced specific
acetate production rate despite a dramatic increase in acetate
production from 3.16 to 14.78 mmol (g-cell h)−1 for the
control strain when the dilution rate changes from 0.1 to 0.4
h−1 (Fig. 4 insert). The flux to ethanol, while it is very
comparable for dilution rates between 0.1 and 0.3 h−1, is
much lower for the ALS strain at higher dilution rates of
0.35 and 0.4 h−1 (Fig. 4 insert). The flux through PFL is
between 30–50% less for the ALS strain, which is also
reflected in the reduced H2 and formate production. The
carbon dioxide evolution rate is less for the ALS strain for
dilution rates of less than about 0.25 h−1, while it approaches that of the control for higher dilution rates.
E. coli have mainly two alternatives for pyruvate utilization: (1) conversion to lactate in a single step via LDH, or
(2) a more complex pathway that splits pyruvate into acetylCoA and formic acid via PFL. As shown in both Tables III
and IV the control strain converts acetyl-CoA into equal
amounts of ethanol and acetate. Such an equimolar split (␯10
≈ ␯11) at the acetyl-CoA branchpoint allows for the generation of sufficient reducing power (␯4 ≈ 2␯10), and in the
742
meantime it allows for the generation of an additional mole
of ATP per mole of glucose fermented (␯ATP/␯1 ≈ 3 mol
ATP per mol glucose) (Tempest and Neijssel, 1987). The
ALS enzyme, as in the batch case, can compete favorably
with native enzymes that consume pyruvate, namely PFL
and LDH. When the ALS is present, the flux through PFL
(␯8) is reduced by almost 2-fold for the whole range of
dilution rates, while the flux through LDH is significantly
lower for D < 0.3. Further reduction of the flux to lactate is
probably infeasible as the cell has to balance its redox by
disposing excess NADH via the LDH reaction.
An important question is how flux partitioning is regulated at the pyruvate branchpoints. According to the Km
values shown in Table V, anaerobically all pyruvate would
Table V.
point.
Apparent Km values of enzymes acting at the pyruvate branch-
Enzyme
Km
(mM)
Temperature
pH
References
PFL
LDH
ALS
2.0
7.2
13.0
30°C
23°C
37°C
7.4–7.8
7.5
7.0
Knappe et al. (1969)
Tarmy and Kaplan (1968)
Holtzclaw (1975)
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 63, NO. 6, JUNE 20, 1999
be channeled preferentially through PFL in the absence of
any superimposed regulation. Besides enzyme regulation at
the transcription level, the catalytic activity of many enzymes is also regulated by reversible binding of “regulator
molecules” (allosteric regulation), which can affect its apparent Km and/or Vmax (Price and Stevens, 1989). Regulation at the E. coli glyoxylate bypass, is an instructive example of allosteric regulation. When the cells are grown on
acetate, the enzymes isocitrate dehydrogenase (ICDH) and
isocitrate lyase (ICL) share a common substrate, isocitrate
(IC), converting it to succinate or glyoxylate, respectively.
What is extraordinary in this case is the fact that the partition between ICDH and ICL is 2:1 (Holms, 1986) despite
the fact that the Km values of the two enzymes differ by a
factor of 75 (8 and 604 mM for ICDH and ICL, respectively). Flux partitioning in this case is achieved by reversible phosphorylation of ICDH which inactivates this enzyme and allows the intracellular concentration of IC to
increase to concentrations that are favorable for ICL (ElMansi et al., 1986).
A mechanism similar to the above is also possible at the
pyruvate branchpoint. Anaerobically, PFL has the highest
affinity for pyruvate, and this is reflected by the high flux
through this enzyme (␯8). The high fluxes through LDH
obtained for D < 0.25 for the control strain are probably a
consequence of higher intracellular pyruvate levels, since
this enzyme is allosterically activated by pyruvate (Tarmy
and Kaplan, 1968a,b). On the other hand, the ability of the
ALS enzyme to effectively compete with both PFL and
LDH will most likely depend on the level of residual acetate
(Brown et al., 1972). Acetate plays a key regulatory role in
organisms that express the pH 6 ALS enzyme, such as Aerobacter aerogens (Johansen et al., 1975) and B. subtilis
(Holtzclaw and Chapman, 1975) preventing the extensive
acidification of their growth environment. At the transcriptional level acetate induces the expression of the ALS (and
other enzymes of the butanediol pathway that redirects pyruvate to neutral species) (Störmer, 1968; Renna et al.,
1993) and it also acts as an activator. Enzymatic studies
have indicated that acetate enhances ALS activity by more
than 40% (Holtzclaw and Chapman, 1975).
Theoretical Calculations of the Biomass Yield on
ATP (YATP)
Early studies on the energetics of microbial growth revealed
a correlation between the amount of biomass formed and the
amount of energy as ATP generated by catabolism. Bauchop and Elsden (1960) reported experiments with bacteria
and yeast grown anaerobically, where they concluded that
the amount of growth was directly proportional to the ATP
that can be derived from catabolism of the carbon source.
The same study introduced the concept of yield of biomass
on ATP, defined as the amount of biomass produced in g
dry weight per mole of ATP (YATP) synthesized from catabolism. The latter quantity can be estimated from biochemical considerations that rely upon knowledge of the
catabolic pathways and the stoichiometry of ATP formation. Anaerobic growth, where ATP synthesis occurs exclusively by substrate level phosphorylation, provides an accurate way to calculate YATP, since the stoichiometry of
oxidative phosphorylation is both difficult to assess and also
may vary with growth conditions (Thauer et al., 1977).
Pirt (1965) derived the following equation that relates the
molar growth yield and the specific growth:
1
ms
1
= max +
Yglu Y glu D
(1)
In the above expression, Yglu (g-cell mol−1 glucose) is the
apparent molar growth yield for glucose, ms is the maintenance coefficient for glucose (mol glucose(g-cell h)−1) and
Ymax
glu is the molar growth yield for glucose corrected for
maintenance. Stouthamer adapted the above expression by
substituting glucose with ATP as shown below:
1
YATP
=
1
max
Y ATP
+
me
D
(2)
where YATP (g-cell mol−1 ATP) is the molar growth yield
for ATP, me is the maintenance coefficient for ATP (mol
ATP (g-cell h)−1) and Ymax
ATP is the molar growth yield for
ATP corrected for maintenance (Stouthamer and Bettenhaussen, 1973).
In their studies, Bauchop and Elsden concluded that the
yields of Streptococcus faecalis, Saccharomyces cerevisiae,
and Zymomonas mobilis per mole of ATP was constant at
about 10.5, leading to the proposal that Ymax
ATP was a biological constant. However, subsequent work with a much wider
variety of organisms has shown Ymax
ATP is not a constant, and
values varying from 4.7 for Z. mobilis to 28.5 for A. aerogens have been reported (Quayle, 1979). On one hand such
calculations have to rely on the precise knowledge of the
fueling pathways, which can be erroneous for certain microbes. On the other hand, an array of studies has shown
that Ymax
ATP can be strongly influenced by a number of factors,
such as the pathways of substrate breakdown (oxidative/
substrate phosphorylation), nature and complexity of the
medium (carbon source, C/N ratio, etc.), the growth temperature, or growth characteristics (specific growth rate,
maintenance, cell components) (Hempfling and Mainzer,
1975; Farmer and Jones, 1976; Mainzer and Hempfling,
1976; Stouthamer, 1977). Literature data (Table VI) illustrate the effects of the growth temperature, carbon source
and oxygen availability on the Ymax
ATP of E. coli.
Equations (1) and (2) can also correlate the calculated
fluxes with the yield coefficients as shown below:
v1 =
vATP =
D
Y max
glu
+ ms
(3)
+ me
(4)
D
max
Y ATP
ARISTIDOU, SAN, AND BENNETT: METABOLIC FLUX ANALYSIS OF E. COLI EXPRESSING B. SUBTILIS ALS
743
Table VI. Effects of temperature, oxygen, and growth substrates on the
E. coli Y max
ATP .
T
(°C)
C-source
Oxygen
Actual
Y max
ATP
Theoreticala
max
YATP
20.3
30
40
30
30
30
30
25
30
37
Glycerol
Glycerol
Glycerol
Acetate
Pyruvate
Galactose
Glucose
Glucose
Glucose
Glucose
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
11.7
12.7
8.4
7.1
8.6
13.5
13.9
13.5
12.3
10.7
29.5
29.5
29.5
12.9
18.6
33.9
33.9
33.9
33.9
33.9
Reference
b
b
b
c
c
c
c
d
d
d
a
Theoretical yields were calculated based on the known energy requirements for the biosynthesis of cell material and assuming that cellular macromolecular composition is 52.4% protein, 16.6% polysaccharide, 15.7%
RNA, 9.4% lipid, and 3.2% D.
b
Farmer (1976a).
c
Farmer (1976b).
d
Mainzer (1976).
where ␯1 corresponds with the net glucose uptake rate and
␯ATP is the net ATP synthesis rate (⳱ 3␯3 + ␯11). The
steady-state conditions of chemostats provide a systematic
way of calculating the yields, with the dilution rate, D,
replacing m in the above equations. The yield coefficients
max
Ymax
glu and YATP can be estimated by plotting ␯1 and ␯ATP vs
the dilution rate, respectively, according to the above equations. For improved accuracy, the values for ms and me are
estimated from a second set of plots of ␯1/D and ␯ATP/D vs
1/D, respectively, i.e. by dividing both sides of Eqs. (3) and
(4) by D (Fieschko and Humphrey, 1983).
The observed yield coefficients for glucose are shown in
Fig. 5A; the “true” yields and the maintenance coefficients
determination are shown in Fig. 5B. Similar to the fluxes
shown in Fig. 4B, the specific glucose uptake rates for the
ALS strain also show quite a different trend from the control
strain, especially at high dilution rates. Two distinct regions
for the control strain can be identified: one at the low dilution rates with a shallower slope and the other at high dilution rates with a much steeper slope. The transition between these two regions appears to occur at around D ⳱
0.25 h−1. These trends are much more pronounced for the
control strain and are less obvious for the ALS strain. Two
estimates for Ymax
glu were performed, and the results of regression analyses are included in Fig. 5B. For D between
0.1 and 0.3 h−1, the control strain has a higher Ymax
glu ; 41.0 vs
32.5 (g-cell mol−1 glucose). The lower maximum glucose
yield for the ALS strain is probably due to the metabolic
burden imposed by the production of the ALS enzyme.
However, the Ymax
glu for the control strain drops precipitously
to 12.7 for dilution rates between 0.3 and 0.4. This decline
is a result of rapid increases in the acetate and ethanol fluxes
at these higher growth rates (Fig. 4 insert). The ALS strain,
on the other hand, is able to keep both acetate and ethanol
at low levels (Fig. 4 insert). Subsequently, Ymax
glu is main-
744
tained at a level similar to that of the lower dilution rates. In
fact, it increases slightly to 38.8. The maintenance coefficients for both systems are estimated from the low dilution
rate data (Fig. 5B insert). Higher dilution rate data are not
included because the contribution to the overall glucose
yield from maintenance becomes much less significant at
these dilution rates and hence may introduce estimation errors. The maintenance coefficients (ms) for the control and
ALS strain are 2.71 and 0.43 (mmol glucose (g-cell h)−1),
respectively. The amount of glucose consumed for maintenance functions is apparently reduced by about 2.28 mmol
of glucose per g-cell for the ALS strain. The enhanced
efficiency of glucose utilization is presumably coupled to
the improved ATP yields discussed below
The observed ATP yield coefficients are shown in Fig.
6A; the “true” yield and the maintenance coefficient determination are shown in Fig. 6B. Similar to the results shown
in Fig. 5B, the net specific ATP synthesis rates for the ALS
strain also show quite a different trend from the control
strain, especially at high dilution rates. Again, two distinct
regions for the control strain can be identified: one at the
low dilution rates with a shallower slope and the other at
high dilution rates with a much steeper slope. Two estimates
for were performed and the results of the regression analysis
are included in Fig. 5B. In contrast to the Ymax
glu , the ALS
−1
strain has a higher Ymax
ATP for D between 0.1 and 0.3 h ; 12.3
−1
vs 11.5 (g-cell mol ATP). The efficiency of the ALS strain
is more apparent at higher dilution rates. While the Ymax
ATP
declined to 8.1 for the ALS strain, the control strain drops
rapidly to 3.20 for dilution rates between 0.3 and 0.4. The
true ATP yield coefficients for the ALS strain is hence more
than twice that of the control. The maintenance coefficients
for both systems are estimated from the low dilution rate
data (Fig. 6B insert). Again, higher dilution rate data are not
being used because the contribution to the overall glucose
yield from maintenance becomes much less significant at
these dilution rates and hence may introduce estimation errors. The maintenance coefficients (me) for the control and
ALS strains are 9.5 and 3.1 (mmol ATP (g-cell h)−1), respectively. The maintenance coefficient for the ALS strain
is only one third that of the control strain.
Assuming that the estimates for me can be extended to the
batch fermentation (Table III), the values for Ymax
ATP is calculated to be in the range of 10.4–12.5 for the control and
10.7–12.9 for the ALS system based on growth rates of
0.25–0.3 h−1. Hence, the values for the ATP yield coefficients estimated from chemostat data are comparable with
those of similar batch experiments.
DISCUSSION
In both batch and continuous experiments, the presence of
the ALS enzyme, significantly reduces the carbon flux to
acetate, and to a smaller extent ethanol at high dilution rates.
Expression of this enzyme without any observed decrease in
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 63, NO. 6, JUNE 20, 1999
Figure 5. (A) Summary of observed glucose yields (Yglu) from anaerobic chemostats. (B) Plot of glucose flux (␯1) vs dilution rate (D), and ␯1/D vs 1/D
(insert) for the determination of “true” biomass yield on glucose and glucose maintenance requirements respectively. Regression analysis using all seven
data points was shown in dotted line.
growth illustrates that both the enzyme and its product are
innocuous to this microorganism. Favorable competition of
the ALS enzyme with PFL and LDH for pyruvate is believed to be a result of allosteric activation of ALS by acetate. The relatively high Km value of ALS for pyruvate is
actually advantageous, because a lower one would have lead
to a drain of pyruvate into this pathway. Moreover, in batch
cultivation induction of this enzyme occurs only when acetate levels exceed a level of 10–20 mM, which is again
favorable to cell growth.
As depicted in Fig. 6A the observed ATP yields are considerably higher for the system expressing the ALS compared with the control strain for all dilution rates examined.
Further analysis of this data indicates this discrepancy is
primarily due to the reduced maintenance requirement for
the ALS strain. As indicated in Fig. 6 the true ATP yield
coefficient for the strain expressing the ALS gene is approximately 7% higher for the low dilution rates and 150%
for the high dilution rates when compared to the control
strain. In addition, the maintenance coefficient for the ALS
ARISTIDOU, SAN, AND BENNETT: METABOLIC FLUX ANALYSIS OF E. COLI EXPRESSING B. SUBTILIS ALS
745
Figure 6. (A) Summary of observed ATP yields (YATP) from anaerobic chemostats. (B) Plot of ATP flux (␯ATP) vs dilution rate (D), and ␯ATP/D vs 1/D
(insert) for the determination of “true” biomass yield on ATP and ATP maintenance requirements respectively. Regression analysis using all seven data
points was shown in dotted line.
strain is only one third that of the control. It clearly indicated that the benefits the cells experienced are far greater
than the burden of synthesizing this extra ALS enzyme. It
has long been established that a fraction of the energy generated in catabolism is used in processes other than net
biomass formation, the so-called maintenance energy (Pirt,
1965). Processes involved may be, among others, turnover
746
of macromolecules, futile cycles and maintaining cellular
homeostasis (Tempest and Neijssel, 1984).
Expression of the ALS enzyme reduces the accumulation
of acetate in the growth medium, and hence the extent of
uncoupling. The overall effect of uncoupling is to cause the
passive diffusion of the undissociated acid into the cytoplasm, and thus reduce the proton motive force. In the ab-
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 63, NO. 6, JUNE 20, 1999
sence of metabolic activity, the values for the intra- and
extracellular pH would be equalized and this would eliminate the ⌬pH component of the proton motive force (Baronofsky et al., 1984; Chen and Bailey, 1993). However, for
viable cells where energy is available, the cell will activate
its homeostatic mechanism so as to maintain a constant
cytoplasmic pH (Booth and Kroll, 1983; Krulwich et al.,
1990). Since homeostasis is an energy consuming process
that contributes to the overall maintenance energy requirements me (Padan et al., 1976), the parent strain would consume a larger portion of its available energy for maintenance purposes. For example, the yeast maintenance energy
requirement was found to be a linear function of the concentration of weak acids present in the medium (benzoate
residual concentration ranging from 0 to 10 mM at D ⳱ 0.1
h−1 and pH at 5.0) (Verduyn et al., 1990). Moreover, it was
also estimated that almost 75% of the total energy generated
was lost due to acid uncoupling before washout (Verduyn et
al., 1990). Therefore, reducing the fluxes to actate and
hence the accumulation of this weak acid in the broth by
expressing the ALS enzyme in E. coli is highly beneficial to
the cell.
This material was supported in part by the National Science
Foundation Grants BCS-9315797 and BES 9411928.
APPENDIX A
Enzymatic Reactions for Anaerobic Growth
The following summarizes the central metabolic reactions
(glycolysis and fermentation) in Escherichia coli grown anaerobically on glucose. Measured quantities (excreted products, carbon substrate, and biomass) are represented in boldface.
Reaction
1
Glucose + PEP → Glucose 6-P + Pyruvate
2
3
Glucose-6-P → Biomass
Glucose 6-P + ATP → 2 Glyceraldehyde 3-P + ADP
a. Glucose 6-P → Fructose 6-P
b. Fructose-6-P + ATP → Fructose 1,6-diP + ADP
c. Fructose 1,6-diP → Dihydroxyacetone-P + Glyceraldehyde 3-P
d. Dihydroxyacetone-P → Glyceraldehyde 3-P
Glyceraldehyde 3-P + NAD+ + Pi + ADP → PEP + H2O + NADH + H+ + ATP
a. Glyceraldehyde 3-P + NAD+ + Pi → Glycerate1,3-diP + NADH + H+
4
5
6
7
8
9
10
11
b. Glycerate 1,3-diP + ADP → Glycerate 3-P + ATP
c. Glycerate 3-P → Glycerate 2-P
d. Glycerate 2-P → PEP + H2O
PEP + ADP → Pyruvate + ATP
PEP + CO2 + NH4+ + 2NADH + Pi + ADP → Succinate + NH3 + 2NAD+ + ATP
a. PEP + CO2 → OAA + PI
b. OAA + NADH + NH4+ → Aspartate + NAD+
c. Aspartate → Fumarate + NH3
d. Fumarate + NADH + Pi + ADP → Succinate + NAD+ + ATP
Pyruvate + NADH → Lactate + NAD+
Pyruvate + HSCoA → Formate + Acetyl-CoA
Formate → CO2 + H2
Acetyl-CoA + 2 NADH + 2 H+ → Ethanol + HSCoA + 2 NAD+
a. Acetyl-CoA + NADH + H+ → Acetaldehyde + HSCoA + NAD+
b. Acetaldehyde + NADH + H+ → Ethanol + NAD+
Acetyl-CoA + Pi + ADP → Acetate + HSCoA + ATP
1. Acetyl-CoA + Pi → Acetyl-P + HSCoA
a. Acetyl-P + ADP → Acetate + ATP
12
2. Pyruvate → Acetoin + 2 CO2
a. Pyruvate → a-Acetolactate + CO2
b. ␣-Acetolactate → Acetoin + CO2
Enzyme(s)
Glucose:PTS enzymes;
Enzyme I, HPr, IIGlc, IIIGlc
Glucose-P isomerase
Phosphofructokinase, PFK
Fructose-diP aldolase
Triose-P isomerase
3-P Glyceraldehyde
dehydrogenase
3-P glycerate kinase
P glycerate mutase
Enolase
Pyruvate kinase
PEP carboxylase
Aspartate aminotransferase
Aspartase
Fumarate reductase
Lactate dehydrogenase
Pyruvate formate-lyase
formate hydrogen-lyase
Aldehyde dehydrogenase
Alcohol dehydrogenase
Acetate phosphotransferase,
PTA
Acetate kinase, ACK, or,
chemical hydrolysis
Acetolactate Synthase, ALS
Acetolactate decarboxylase
or chemical hydrolysis
ARISTIDOU, SAN, AND BENNETT: METABOLIC FLUX ANALYSIS OF E. COLI EXPRESSING B. SUBTILIS ALS
747
Derivation of NADH and ATP Production rate
(a) Batch
A. Oxidoreductive (NAD+/NADH) Balances
Redox reaction
Participating reactions
Both the biomass and byproducts accumulates in the bioreactor and their concentrations vary with time, i.e.,
v4
Glycerate 1,3-diP
1. Glyceraldehyde 3-P + NAD+ + Pi →
+ NADH + H+
v6
2. OAA + NADH + NH4+ →
Aspartate + NAD+
v6
3. Fumarate + NADH + Pi + ADP →
Succinate + NAD+
+ ATP
v7
4. Pyruvate + NADH →
Lactate + NAD+
v 10
5. Acetyl-CoA + NADH + H + →
Acetaldehyde +
HSCoA + NAD+
v10
6. Acetaldehyde + NADH + H+ →
Ethanol + NAD+
dX
⫽0
dt
and
dCi
⫽0
dt
Two approximation techniques are usually employed for
estimating the specific synthesis rate, ri.
1. Polynomial fit which involves the approximation of the
concentration profiles by polynomial functions; specific
growth and synthesis rates are then calculated from these
time profiles.
2. Time averaged concentrations in which the specific
rates between two successive sampling periods (t and t+⌬t)
are estimated based on the average cell density (log mean):
Overall Balance: rNADH ⳱ v4 − 2v6 − v7 − 2v10
wi = wi
w共t + ⌬t兲 − w共t兲
x⌬t
B. Energy (ATP) Balances
Net ATP synthesis: this is calculated as the sum of all fluxes
that are coupled to ATP synthesis.
Participating reactions
where,
1.
2.
3.
4.
v3
Fructose 1,6-diP + ADP
Fructose-6-P + ATP →
v4
Glycerate 1,3-diP + ATP →
Glycerate 3-P + ATP
v5
PEP + ADP → Pyruvate + ATP
v6
Fumarate + NADH + Pi + ADP →
Succinate + NAD+
+ ATP
v11
5. Acetyl-P + ADP →
Acetate + ATP
It can be shown that the above equation provides a good
estimate when the specific synthesis rate and the specific
growth rate are constant during the two sampling points.
These conditions are generally assumed to be met under
balanced growth.
Overall Balance: rATP ⳱ − v3 + v4 + v5 + v6 + v11 ⳱ 3v3
+ v11
(b) Chemostat
APPENDIX B
x=
x共t + ⌬t兲 − x共t兲
= log mean cell density.
ln共x共t + ⌬t兲 Ⲑ x共t兲兲
The steady state of the continuous culture provides an extremely convenient way to determine the specific conversion rates. At steady state,
dX dCi
=
=0
dt
dt
Specific Rate Determination for Batch vs
Chemostat Cultivations
The specific net conversion rate, ri, is determined experimentally by measuring the rate of accumulation of extracellular metabolites. The following is a general expression
for the rate of change of concentration in biomass and metabolite i:
dCi
dX
= ␮X − DX and
= ri X − DCi
dt
dt
−1
where Ci (mmol L ) is the extracellular concentration of
metabolite i, X (g L−1) is the biomass concentration, ␮ (h−1)
is the specific growth rate and D is the dilution (h−1). The
dilution is given by Fl/Vr with Fl and Vr denote the nutrient
flow rate (L h−1) and the fermentor liquid volume, respectively, and is equal to zero for batch reactor. The analysis
for batch and chemostat cultures will be discussed separately.
748
⇒ ␮=D
and ri = D
Ci
X
Since both X and Ci are time invariant and thus can be
measured several times once a steady state is reached. These
repeated measurements at steady states should improve the
quality of the data. The above expression can be used for all
metabolites excreted into the fermentation broth. A similar
approach can also be applied to the gaseous products CO2
and H2 (Aristidou 1994).
References
Aristidou AA. 1994. Application of Metabolic and Biochemical Engineering Techniques for the Enhancement of Recombinant Protein Production in Escherichia coli. Ph.D. Thesis, Department of Chemical Engineering, Rice University, Houston, TX.
Aristidou AA, Bennett GN, San K-Y. 1994. Modification of the central
metabolic pathways of Escherichia coli to reduce acetate accumulation
by heterologous expression of the Bacillus subtilis acetolactate synthase gene. Biotechnol Bioeng 44(8):944–951.
Aristidou AA, San K-Y, Bennett GN. 1995. Metabolic engineering of
Escherichia coli to enhance recombinant protein production through
acetate reduction. Biotechnol Prog 11(4):475–478.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 63, NO. 6, JUNE 20, 1999
Baronofsky JJ, Schreurs WJA, Kashket ER. 1984. Uncoupling by acetic
acid limits growth of and acetogenesis by Clostridium thermoaceticum. App Environ Microbiol 48:1134–1139.
Bauchop T, Elsden SR. 1960. The growth of microorganisms in relation to
their energy supply. J Gen Microbiol 23:457–469.
Blackwood AC, Neish AC, Ledingham GA. 1956. Dissimilation of glucose
at controlled pH values by pigmented and non-pigmented strains of E.
coli. J Bacteriol 72:497–499.
Booth IR, Kroll RG. 1983. Regulation of cytoplasmic pH (pHi) in bacteria
and its relationship to metabolism. Biochem Soc Trans 11(1):70–72.
Brown TDK, Pereira CRS, Störmer FC. 1972. Studies of the acetate kinase–phosphotransacetylase and the butanediol-forming systems in
Aerobacter aerogens. J Bacteriol 121(3):1106–1111.
Chang ACY, Cohen SN. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the p15A
cryptic miniplasmid. J Bacteriol 134(3):1141–1156.
Chen R, Bailey JE. 1993. Observations of aerobic, growing Escherichia
coli metabolism using an on-line nuclear magnetic resonance spectroscopy system. Biotechnol Bioeng 42(2):215–221.
Cherrington CA, Hinton M, Chopra I. 1990. Effect of short-chain organic
acids on macromolecular synthesis in Escherichia coli. J Appl Bacteriol 68:69–74.
Cherrington CA, Hinton M, Pearson GR, Chopra I. 1991. Short-chain
organic acids at pH 5.0 kill Escherichia coli and Salmonella spp.
without causing membrane perturbation. J Appl Bacteriol 70:161–165.
Dawson PSS. 1984. Continuous cultivation of microorganisms. CRC Crit
Rev Biotechnol 2(4):315–373.
Diaz-Ricci JC, Hiltzmann B, Rinas U, Bailey JE. 1990. Comparative studies of glucose catabolism by Escherichia coli grown in complex medium under aerobic and anaerobic conditions. Biotechnol Prog 6:
326–332.
Doelle HW. 1975. Regulation of carbohydrate metabolism in facultative
anaerobic bacteria (Pasteur and Crabtree effects), pp. 609–691. In:
Bacterial Metabolism. London: Academic Press.
El-Mansi EMT, Nimmo HG, Holms WH. 1986. Pyruvate metabolism and
the phosphorylation state if isocitrate dehydrogenase in Escherichia
coli. J Gen Microbiol 132:797–806.
Farmer IS, Jones CW. 1976. The effect of temperature on the molar growth
yield and maintenance requirement of Escherichia coli W during aerobic growth in continuous culture. FEBS Lett 67(3):359–363.
Fieschko J, Humphrey AE. 1983. Statistical analysis in the estimation of
maintenance and true growth yield coefficients. Biotechnol Bioeng
26:394.
Hempfling WP, Mainzer SE. 1975. Effects of varying the carbon source
limiting growth on yield and maintenance characteristics of Escherichia coli in continuous culture. J Bacteriol 123(3):1076–1087.
Farmer IS, Jones CW. 1976. The energetics of Escherichia coli during
aerobic growth in continuous culture. Eur J Biochem 67:115–122.
Holms WH. 1986. The Central Metabolic Pathways of Escherichia coli:
Relationship between Flux and Control at a Branchpoint, Efficiency of
Conversion to Biomass, and Excretion of Acetate. Curr Top Cell Reg
28:69–105.
Holtzclaw WD, Chapman LF. 1975. Degrative acetolactate synthase of
Bacillus subtilis: Purification and properties. J Bacteriol 121:917–922.
Ingram LO, Conway T. 1988. Expression of different levels of ethanolgenic enzymes from Zymomonas mobilis in recombinant strains of
Escherichia coli. Appl Environ Microbiol 54(2):397–404.
Jensen EB, Carlsen S. 1990. Production of recombinant human growth
hormone in Escherichia coli: Expression of different precursors and
physiological effects of glucose, acetate, and salts. Biotechnol Bioeng
36:1–11.
Johansen L, Bryn K, Störmer FC. 1975. Physiological and biochemical role
of the butanediol pathway in Aerobacter (Enterobacter) aerogens. J
Bacteriol 123:1124–1130.
Knappe JH, Balschkowski P, Grobner P, Schmitt T. 1974. Pyruvate formate-lyase of Escherichia coli: The acetyl-enzyme intermediate. Eur J
Biochem 50:253–263.
Krulwich TA, Quirk PG, Guffanti AA. 1990. Uncoupler-resistant mutants
of bacteria. Microbiol Rev 54(1):52–65.
Mainzer SE, Hempfling WP. 1976. Effect of growth temperature on yield
and maintenance during glucose-limited continuous culture of Escherichia coli. J Bacteriol 126(1):251–256.
Majewski RA, Domach MM. 1990. Simple constrained optimization view
of acetate overflow in Escherichia coli. Biotechnol Bioeng 35:
732–738.
Meyer H-P, Leist C, Fiechter A. 1984. Acetate formation in continuous
culture of Escherichia coli K12 D1 on defined and complex media. J
Biotechnol 1:335–358.
Padan E, Zilbertstein D, Rottenberg H. 1976. The proton electrochemical
gradient in Escherichia coli cells. Eur J Biochem 63:533–541.
Pascal MC, Chippaux M, Jaoudé AA, Blaschkowski HP, Knappe J. 1981.
Mutants of Escherichia coli K12 with defects in anaerobic pyruvate
metabolism. J Gen Microbiol 124:35–42.
Pecher A, Zinoni F, Jatisatienr C, Wirth H, Hennecke H, Böck A. 1983. On
the redox control of synthesis of anaerobically induced enzymes in
enterobacteriaceae. Arch Microbiol 136:131–136.
Pirt SJ. 1965. The maintenance energy of bacteria in growing cultures. Proc
R Soc Lond B 163:224–231.
Price NC, Stevens L. 1989. Fundamentals of Enzymology. Oxford: Oxford
University Press.
Quayle JR. 1979. Microbial Biochemistry, pp. 381. In: Kornberg HL,
Phillips DC, editors. International Review of Biochemistry. Baltimore,
MD: University Park Press.
Renna MC, Najimudin N, Winik LR, Zahler SA. 1993. Regulation of the
Bacillus subtilis alsS, alsD, and alsR genes involved in postexponential-phase production of acetoin. J Bacteriol 175(12):
3863–3875.
Stephanopoulos G, Vallino JJ. 1991. Network Rigidity and Metabolic Engineering in Metabolite Overproduction. Science 252(5013):1675.
Störmer FC. 1968. Evidence for induction of the 2,3-butanediol-forming
enzymes in Aerobacter aerogenes. FEBS Lett 2(1):36–38.
Stouthamer AH. 1977. Energetic aspects of the growth of micro-organisms,
pp. 285–315. In: Haddock BA, Hamilton WA, editors. Microbial Energetics: Twenty-Seventh Symposium of the Society for General Microbiology. Cambridge: Cambridge University Press.
Stouthamer AH, Bettenhaussen CW. 1973. Utilization of energy for growth
and maintenance in continuous and batch cultures of micro-organisms.
A re-evaluation of the method for the determination of ATP production
by measuring molar growth yields. Biochim Biophys Acta 301:53–70.
Tarmy EM, Kaplan NO. 1968a. Chemical characterization of D-lactate
dehydrogenase from Escherichia coli B. J Biol Chem 243:2579–2586.
Tarmy EM, Kaplan NO. 1968b. Kinetics of Escherichia coli B D-lactate
dehydrogenase and evidence for pyruvate controlled change in conformation. J Biol Chem 243:2587–2596.
Tempest DW, Neijssel OM. 1984. The status of YATP and maintenance
energy as a biologically interpretable phenomena. Annu Rev Microbiol 38:459–486.
Tempest DW, Neijssel OM. 1987. Growth yield and energy distribution,
pp. 797–806. In: Neidhardt FC, editor. Escherichia coli and Salmonella typhimurium. Washington, DC: American Society for Microbiology.
Thauer RK, Jungermann K, Decker K. 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180.
Verduyn C, Postma E, Scheffers WA, van Dijken JP. 1990. Energetics of
Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J Gen Microbiol 136:405–412.
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