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ARTICLE
Engineering Butanol-Tolerance in Escherichia coli
With Artificial Transcription Factor Libraries
Ju Young Lee, Kyung Seok Yang, Su A Jang, Bong Hyun Sung, Sun Chang Kim
Department of Biological Sciences, Korea Advanced Institute of Science and Technology,
373-1 Guseong-dong Yuseong-gu, Daejeon 305-701, Korea, telephone: 82-42-350-2619;
fax: 82-42-350-2610; e-mail: sunkim@kaist.ac.kr
Received 26 May 2010; revision received 10 October 2010; accepted 18 October 2010
Published online 28 October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.22989
ABSTRACT: Escherichia coli has been explored as a host for
butanol production because of its many advantages such as a
fast growth and easy genetic manipulation. Butanol toxicity,
however, is a major concern in the biobutanol production
with E. coli. In particular, E. coli growth is severely inhibited
by butanol, being almost completely stopped by 1% (vol/
vol) butanol. Here we developed a new method to increase
the butanol-tolerance of E. coli with artificial transcription
factor (ATF) libraries which consist of zinc finger (ZF)
DNA-binding proteins and an E. coli cyclic AMP receptor
protein (CRP). Using these ATFs, we selected a butanol-
tolerant E. coli which can tolerate up to 1.5% (vol/vol)
butanol, with a concomitant increase in heat resistance.
We also identified genes of E. coli that are associated with
the butanol-tolerance. These results show that E. coli can be
engineered as a promising host for high-yield butanol
production.
Biotechnol. Bioeng. 2010;xxx: xxx–xxx.
!2010 Wiley Periodicals, Inc.
KEYWORDS: butanol-tolerance; heat resistance; artificial
transcription factor; butanol production
Introduction
Concerns about a shortage of available fossil fuels in the
future, coupled with environmental problems resulting
from their extraction, refining and use, have spurred
increased efforts to synthesize biofuels from renewable
resources. Among biofuels, butanol has recently received
special attention as a renewable resource fuel alternative on
the basis of a number of attractive attributes, including its
comparable energy content to gasoline, low hygroscopicity,
and ability to be fermented from a wide variety of carbon
sources (Inui et al., 2008). Butanol can also be blended with
gasoline or added to diesel fuel to reduce viscosity, and is
compatible with existing supply infrastructures (Atsumi
et al., 2007; Rutherford et al., 2010). Typically, butanol is
produced by acetone–butanol–ethanol (ABE) fermentation
using Clostridium species (George et al., 1983; Lin and
Blaschek, 1983). Clostridium, however, is a strict anaerobe
and has a significantly slow growth rate, leading to low
butanol productivity. Furthermore, the relatively unknown
genetic system and complex physiology of Clostridium
present difficulties in engineering its metabolic pathway for
the efficient production of butanol.
In response to this, Escherichia coli has been engineered as
an alternative host for butanol production by introducing a
butanol production pathway (Atsumi et al., 2007, 2008; Inui
et al., 2008; Nielsen et al., 2009), because it is easy to
manipulate genetically and grows fast, allowing for a flexible
and economical process design for large-scale production
(Ingram et al., 1999; Khosla and Keasling, 2003). However,
in biobutanol production using E. coli, butanol toxicity is a
major concern (Alper et al., 2006; Atsumi et al., 2008),
because E. coli growth is severely inhibited by butanol, being
almost completely stopped by 1% (vol/vol) butanol (Atsumi
et al., 2008). This lack of butanol-tolerance of E. coli has
spurred research on the development of E. coli strains with
improved butanol-tolerance. However, in general, butanol-
tolerant phenotypes are difficult to engineer, because stress
conditions such as exposure to organic solvents elicit
multigenic responses coordinated at the transcriptomic and
proteomic levels (Tomas et al., 2004).
Industrial strains with tolerance to final fuel products as
well as other relevant toxic compounds have traditionally
been obtained through adaptation and selection (Yomano et
al., 1998). However, since natural adaptation is time- and
Additional supporting information may be found in the online version of this article.
Correspondence to: S.C. Kim
Contract grant sponsor: Ministry of Education, Science and Technology of Korea
Contract grant number: MG08-0204-1-0
Contract grant sponsor: Ministry of Education, Science and Technology of Korea
Contract grant number: M10748222314-08N4800-31410
Contract grant sponsor: Basic Program of the Korea Science and Engineering
Foundation
Contract grant number: R01-2008-000-20559-0
Contract grant sponsor: Korean Ministry of Knowledge Economy
Contract grant number: N02071165
!2010 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2010 1
resource-intensive, several mutagens have been successfully
used to introduce diversity in the population of strains
and accelerate strain improvement (Parekh et al., 2000).
Recently, in recognition of the more direct mapping
between transcriptome and phenotype, efforts in strain
improvement have focused on direct manipulation of the
transcript profile. Global transcription machinery engineer-
ing (gTME) has been applied to introduce transcriptional-
level modifications (Alper and Stephanopoulos, 2007; Alper
et al., 2006). We recently developed novel artificial
transcription factors (ATFs) composed of zinc finger (ZF)
DNA-binding proteins, with distinct specificities, fused to
an E. coli cyclic AMP receptor protein (CRP). We also
demonstrated the use of ATF libraries to elicit multiple
simultaneous transcriptional-level modifications and thus
induce phenotypic variations in E. coli (Lee et al., 2008).
Here, we describe their application to improve butanol-
tolerance in E. coli to render it more suitable for butanol
production. With the ATF libraries, we selected a butanol-
tolerant E. coli that can withstand up to 1.5% (vol/vol)
butanol, with a concomitant increase in heat resistance.
Genes associated with butanol-tolerance were also identified
and characterized.
Materials and Methods
Strains and Plasmids
E. coli K-12 MG1655 was used for all of the experiments.
Plasmid pETtac containing a tac promoter was used for
expression of the ATF libraries, as previously described (Lee
et al., 2008). Plasmid pACYCtac (Lee et al., 2008) was used
for expression of genes related to the butanol-tolerant
phenotype. For complementary assays, the genes related
to the butanol-tolerant phenotype, sdhCDAB,flu,ybgD,
and glpC, were amplified with a polymerase chain reaction
(PCR) from MG1655 genomic DNA, and the amplified
genes were cloned into pETtac and pACYCtac, individually
or together, producing pETtac-sdhCDAB, pETtac-flu,
pETtac-ybgD, pETtac-glpC, pETtac-sdhCDAB/ybgD, and
pACYCtac-flu/glpC, respectively.
Eliciting a Butanol-Tolerant Phenotype With ATF
Libraries
By randomly assembling 40 different types of ZFs, we
previously constructed more than 6.4 !10
4
ATFs, each
consisting of 3 ZF DNA-binding domains and a CRP D1
(residues 1–180) effector domain (Lee et al., 2008). E. coli
MG1655 was electrotransformed with 10 ng of constructed
ATF libraries (the pETtac-CRP D1-3ZF libraries), with a
transformation efficiency of 1 !10
8
cfu/mg DNA. The
resulting 10
6
transformants that exceeded the complexities
of the ATF libraries were analyzed for butanol-tolerance
upon ATF expression.
Cells with improved butanol-tolerance were screened as
described previously (Alper and Stephanopoulos, 2007)
with some modifications. Briefly, ATF transformants were
cultured in LB medium with 0.5 mM IPTG for 3 h at 308C to
induce ATF expression. Cells were then subcultured by
10-fold dilution into LB medium containing butanol in a
range of 1–2% (vol/vol) and 0.5 mM IPTG. After 24 h of
incubation at 308C, cells were plated on LB agar plates and
incubated overnight at 308C. The surviving cells were
screened again for the butanol-tolerant phenotype and cell
growth was assessed. The cell growth profiles were examined
in 100 mL of LB medium with varying concentrations of
butanol (as indicated in Figs. 1–3) and 0.5 mM IPTG at
308C, starting from an initial optical density (OD) at 600 nm
of 0.1. To elucidate the effect of iron ions on the butanol-
tolerance of cells, a medium was supplemented with 0.075 g/
L of FeSO
4
(Shiloach and Fass, 2005). The growth of cells
was monitored by measuring the cell density at OD
600
.
Plasmids that encoded ATFs were rescued from the
selected cells that showed butanol-tolerant phenotypes,
and the ATF sequences of the rescued plasmids were
identified by DNA sequencing. The rescued plasmids with a
confirmed ATF were retransformed back into wild-type
E. coli to examine whether the plasmids induce the same
phenotypic change or not.
Quantification of Intracellular Adenosine Triphosphate
(ATP)
E. coli cells were cultured in LB medium with 0.5 mM IPTG
with or without butanol (1%, vol/vol) at 308C. The ATP was
Figure 1. A butanol-tolerant phenotype induced by ATF libraries. Cells were
grown in LB medium with varying concentrations of butanol (1–1.6%, vol/vol), starting
from an initial OD
600
of 0.1. After 24 h of incubation at 308C, the cell growth was
monitored by measuring the cell density at OD
600
. Error bars denote the standard
deviation (SD) from the mean of three independent experiments. C, the wild-type E. coli
transformed with a control plasmid. BT, the butanol-tolerant cells expressing BT ATF.
After the first round of phenotypic screening, the phenotypic changes were confirmed
by plasmid rescue, sequence analysis, and retransformation.
2Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2010
extracted from the cells in the exponential and stationary
phase of growth (6 and 24 h after inoculation, respectively)
and the concentration of ATP was determined with an ATP
bioluminescence assay kit HS II (Roche Applied Science,
Mannheim, Germany) according to the manufacturer’s
protocol. Luminescence was monitored with a microplate
luminometer (Microlumat LB 96 P, EG&G Berthold, Bad
Wildbad, Germany).
DNA Microarray Analysis of Butanol-Tolerant Cells
E. coli cells transformed with either a plasmid encoding the
ATF that induced a butanol-tolerant phenotype (BT ATF)
or the pETtac plasmid only were cultured in LB medium
with 0.5 mM IPTG at 308C. Total RNA was isolated from
cells grown to an exponential phase with RNeasy mini kit
(QIAGEN, Valencia, CA) and RNAprotect Bacteria Reagent
(QIAGEN), and the RNA preparations were subjected to
reverse transcription. cDNA labeled with either Cy3-dUTP
(C, wild-type E. coli transformed with a control plasmid) or
Cy5-dUTP (BT, cells transformed with a plasmid encoding
the BT ATF) was synthesized from each preparation of
the total RNA by random priming. Labeled cDNA probes
were purified and hybridized to a DNA microarray slide
(TwinChip
TM
E. coli chip, Digital Genomics, Seoul, Korea)
that contained the complete E. coli genome. The slides were
scanned by Scanarray Lite (Packard Bioscience, Boston,
MA) and analyzed by GenePix Pro 3.0 software (Axon
Instrument, Union City, CA).
Heat Resistance of the Butanol-Tolerant Cells
BT butanol-tolerant cells were grown at 308C to a stationary
phase (OD
600
¼3) in LB medium with 0.5 mM IPTG. Cells
were then incubated for 2 h at 558C, and 2 mL of 10-fold
serial dilutions of the cells was spotted on LB agar plates,
as previously described (Lee et al., 2008). The growth
Figure 2. Growth profiles of the butanol-tolerant cells (BT) in LB medium
(100 mL) containing 1 (A) and 1.5% (vol/vol) butanol (B), respectively. The ODs were
measured with a spectrophotometer at 600 nm at different time intervals. The growth
experiments were performed in triplicate. Error bars represent SD. C, the wild-type
E. coli transformed with a control plasmid. BT, the butanol-tolerant cells expressing
BT ATF.
Figure 3. Identification of target genes associated with butanol-tolerance.
A: Analysis of butanol-tolerance by overexpression of the sdhCDAB,flu,ybgD,
or/and glpC genes. Cells were grown in LB medium with varying concentrations of
butanol (1–1.6%, vol/vol), starting from an initial OD
600
of 0.1. B: Effect of iron ions on
butanol-tolerance. Cells were grown under the same conditions described above,
except that the medium was supplemented with 0.075 g/L of FeSO
4
. After 24 h of
incubation at 308C, the cell growth was monitored by measuring the cell density
at OD
600
. Inset graph shows fold improvements of growth yields which are compared
with cells grown in LB medium without iron ions. Error bars denote SD from the mean
of three independent experiments. C, the wild-type E. coli transformed with a control
plasmid. BT, the butanol-tolerant cells expressing BT ATF. sdhCDAB,flu,ybgD, and
glpC, cells overexpressing sdhCDAB,flu,ybgD, and glpC, respectively. sþfþyþg,
cells co-expressing sdhCDAB,flu,ybgD, and glpC.
Lee et al.: Engineering Butanol-Tolerance in E. coli 3
Biotechnology and Bioengineering
of cells was monitored after incubation for 12 h at 308C.
The heat resistance of sdhCDAB-, flu-, ybgD-, or/and
glpC-overexpressing cells was also analyzed as described
above.
Results and Discussion
Eliciting a Butanol-Tolerant Phenotype With
ATF Libraries
We introduced ATF libraries constructed with a high-copy
plasmid into E. coli and allowed ATFs to be constitutively
expressed in order to screen the transformed bacteria for a
butanol-tolerant phenotype.
To screen for cells with improved butanol-tolerance, ATF
transformants were cultured in LB medium containing
butanol in a range of 1–2% (vol/vol). Among 10
6
ATF
transformants screened, 75 ATF transformants survived in
LB medium containing 1.5% (vol/vol) butanol, while
surviving cells were not obtained at higher butanol
concentrations (>1.5%). The ATF transformants surviving
at 1.5% (vol/vol) butanol were individually analyzed for
their butanol-tolerance. After confirming that an improved
butanol-tolerant phenotype was indeed conferred by an ATF
(through retransformation into wild-type E. coli with
each ATF plasmid rescued from the selected cells), the
transformant with the most improved butanol-tolerance
(BT) was selected for further study. The BT transformant
expressed BT ATF, which was composed of a CRP D1
(residues 1–180) and three ZFs (Z11, Z4, and Z23, ordered
in the N- to C-terminal direction) (Lee et al., 2008). The
putative binding site for the BT ATF was inferred to be 50-
GG(G/A) A(C/G/A)(A/C/G) (A/G/C)A(A/G/C)-30from
published literature (Bae et al., 2003; Blancafort et al.,
2004; Sera and Uranga, 2002).
To determine the ability of cells to survive and/or grow at
different concentrations of butanol, BT butanol-tolerant
cells and wild-type cells were grown in LB medium with
varying concentrations of butanol at 308C for 24 h and cell
growth was monitored by measuring the cell density
at OD
600
(Figs. 1 and 2). The growth of wild-type cells
was severely inhibited at 1% (vol/vol) butanol. However, the
BT butanol-tolerant cells could tolerate up to 1.5% (vol/vol)
butanol. In particular, the BT butanol-tolerant cells grew
$24- and $9-fold faster with up to $6- and $3-fold higher
OD than wild-type cells in LB medium containing 1% and
1.5% (vol/vol) butanol, respectively (Fig. 2). In the absence
of butanol stress, the wild-type and BT butanol-tolerant cells
exhibited a similar growth pattern.
It is known that butanol increases the fluidity of cellular
membranes and disrupts the activity of membrane-
associated proteins (such as glucose uptake) (Bowles and
Ellefson, 1985). It was also reported that when bacteria are
exposed to butanol, the ratio of saturated to unsaturated
fatty acids incorporated in the membrane increases,
presumably to compensate for the increased membrane
fluidity imposed by butanol (Baer et al., 1987; Lepage et al.,
1987; Vollherbst-Schneck et al., 1984). However, the fatty
acid composition of our BT butanol-tolerant cells was very
similar to that of wild-type cells (Supplementary Table 1)
and a similar inhibition of glucose uptake by butanol was
observed in both the BT butanol-tolerant and wild-type cells
(Supplementary Table 2). This implies that the BT ATF did
not negate the toxic effect of butanol through a decrease in
membrane fluidity caused by changes of the fatty acid
composition or active glucose uptake.
In addition, it has been reported that butanol lowers
intracellular levels of ATP, which in turn leads to inhibition
of cell growth (Bowles and Ellefson, 1985). Therefore, we
measured intracellular ATP concentration in both the wild-
type and BT butanol-tolerant cells grown at 1% (vol/vol)
butanol (Table I), and a significant growth difference was
observed between the wild-type and BT butanol-tolerant
cells. Butanol lowered the intracellular level of ATP in both
strains, but to a lesser extent in the BT butanol-tolerant cells
than in the wild-type cells. The ATP concentration in the BT
butanol-tolerant cells was $1.6-fold lower under butanol
stress (1% [vol/vol] butanol) than in the absence of butanol
stress at an exponential growth phase (6 h after inoculation),
whereas the ATP concentration in the wild-type cells was
lowered $3.3-fold. It appears that a high intracellular ATP
concentration may contribute to constitution of a butanol-
tolerant phenotype to compensate the damage caused by
butanol in BT butanol-tolerant cells, while the actual
mechanism remains to be further studied.
Table I. Cellular concentration of ATP in the butanol-tolerant cells (BT).
Strain
Intracellular ATP (mmol/g DCW)
Without butanol With 1% (vol/vol) butanol
Exponential phase Stationary phase Exponential phase Stationary phase
C 6.059 %0.666 1.794 %0.166 1.815 %0.179 0.853 %0.104
BT 5.324 %0.541 1.971 %0.166 3.235 %0.333 0.853 %0.062
For determination of intracellular ATP concentration, cells were grown in LB medium with or without
butanol (1%, vol/vol) at 308C. The ATP was extracted from the cells in the exponential and stationary growth
phases (6 and 24 h after inoculation, respectively) and measured by luciferase-driven bioluminescence. Values
represent an average of three independent experiments with SD. C, the wild-type E. coli transformed with a
control plasmid and BT, the butanol-tolerant cells expressing BT ATF.
4Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2010
Identification of Genes Associated With
Butanol-Tolerance
A DNA microarray-based transcriptional analysis of the BT
butanol-tolerant cells was performed in order to understand
the transcriptional responses of butanol-tolerance. The
transcriptomic analysis was performed with RNAs isolated
from cells grown in the absence of butanol to assess the effect
of the BT ATF on transcription (Alper et al., 2006).
Transcription profiling of the BT butanol-tolerant cells
revealed that the BT ATF elicited various levels of global
transcription alteration. Specifically, a total of 284 genes
(162 up-regulated and 122 down-regulated) were differen-
tially expressed in the transformant with the BT ATF by
more than 2-fold compared to the control (Supplementary
Tables 3 and 4). Highly up-regulated genes included those
required for energy metabolism, cell envelop biogenesis, and
cell motility, together with many unknown genes. Down-
regulated genes included genes encoding iron-using
proteins, genes involved in amino acid and inorganic ion
transports, and genes of unknown function. However, genes
involved in ATP production were not found to be
differentially up- or down-regulated in the BT butanol-
tolerant cells relative to the control cells despite their high
intracellular ATP concentration; these genes included genes
that encoded ATP synthase and glycogen synthase, genes
involved in de novo biosynthesis of pyrimidine ribonucleo-
tides, and genes of the phosphotransferase system. It appears
that the BT ATF does not affect the ATP synthesis machinery
but rather it affects other cellular functions such as
membrane stability or an indirect energy metabolism for
ATP production, resulting in maintenance of a high
intracellular ATP concentration.
It is known that overexpression of heat shock proteins,
which are expressed in response to diverse environmental
stresses such as heat shock, organic solvents, and osmotic
and oxidative stresses, stabilizes proteins and cellular
structures and thus makes possible prolonged cellular
metabolism and increased butanol-tolerance (Tomas et al.,
2004). Interestingly, however, the expression of these stress-
responsive genes (except for glpC involved in resistance to
organic solvent stress) was not significantly altered in the BT
butanol-tolerant cells relative to the wild-type cells, even
though hundreds of genes were differentially expressed by
the BT ATF. These results suggest that the BT ATF may cause
genetic perturbations in conferring BT that are different
from those induced by exposure to environmental stress
conditions.
Among many genes up-regulated in the BT butanol-
tolerant cells, sdhCDAB,flu, and ybgD genes showed the
largest increases in gene expression relative to the wild-
type cells. Recently, sdhCDAB, which is involved in an
oxidative stress response, was also reported to be up-
regulated during butanol stress (Rutherford et al., 2010).
Therefore, sdhCDAB,flu, and ybgD were selected for further
study; glpC which is known to be involved in organic solvent
tolerance (Shimizu et al., 2005), was also selected (Table II).
In addition, when we examined the regulatory regions of
Table II. Genes associated with butanol-tolerance.
Gene Function
a
The putative BT ATF
bindings site
b
(50–30)
Log
2
fold-change
c
Increased expression
sdhC Succinate dehydrogenase membrane protein
&246
ggggaaaac
&238
4.7
sdhD Succinate dehydrogenase membrane protein
&246
ggggaaaac
&238
4.4
flu Antigen 43 phase-variable biofilm formation autotransporter
&127
gggaacaac
&119
4.3
sdhA Succinate dehydrogenase flavoprotein
&246
ggggaaaac
&238
4.1
ybgD Predicted fimbrial-like adhesin protein
&240
gcaacccaa
&232
4
sdhB Succinate dehydrogenase iron-sulfur protein
&246
ggggaaaac
&238
3.6
glpC sn-glycerol-3-phosphate dehydrogenase
&2902
ggtaaaaaa
&2894
1.6
Decreased expression
fhuF Ferric iron reductase
&56
cgggtaaac
&48
&2.2
fhuA Ferrichrome outer membrane transporter
&280
ggtaaaata
&272
&2.1
cirA Ferric iron-catecholate outer membrane transporter
&220
ggatataaa
&212
&1.5
fepD Ferric enterobactin ABC transporter
&219
ccgagcaaa
&211
&1.5
fhuC Iron-hydroxamate transporter subunit
&280
ggtaaaata
&272
&1.4
fiu Predicted iron outer membrane transporter
&149
gatagaaaa
&141
&1.3
fes Enterobactin esterase
&341
gggaatgaa
&333
&1.3
fecI RNA polymerase, sigma 19 factor
&85
tggaaacaa
&77
&1.2
fepG Ferric enterobactin ABC transporter
&219
ccgagcaaa
&211
&1.2
bfd Bacterioferritin-associated ferredoxin
&175
gcaataaat
&167
&1.2
fepC Ferric enterobactin ABC transporter
&219
ccgagcaaa
&211
&1.1
fepB Ferric enterobactin ABC transporter
&70
gggacggat
&62
&1
tonB Membrane spanning protein in TonB–ExbB–ExbD complex
&223
gggcacaac
&215
&1
a
From the EcoCyc database (http://ecocyc.org).
b
The putative BT ATF binding sites present in the regulatory regions of genes associated with butanol-tolerance. The nucleotide þ1 is the A of the ATG-
translation initiation codon.
c
Log
2
fold-change in gene expression between the BT butanol-tolerant cells and wild-type E. coli containing a control plasmid that lacks an ATF (average of
duplicate experiments).
Lee et al.: Engineering Butanol-Tolerance in E. coli 5
Biotechnology and Bioengineering
sdhCDAB,flu,ybgD, and glpC, we found that they housed
putative binding sites for the BT ATF in their regulatory
regions (Table II).
To determine the functional relevance of the sdhCDAB,
flu,ybgD, and glpC genes to the butanol-tolerant phenotype
in E. coli, we examined the effect of overexpression of these
genes on the butanol-tolerance of E. coli (Fig. 3). When the
sdhCDAB,flu, or ybgD gene was individually overexpressed
in wild-type cells, the resulting cells were more resistant to
butanol stress, with some variations than wild-type cells
under the present butanol-stress conditions, although they
were much less butanol-tolerant than the BT butanol-
tolerant cells. Among these three cells, the sdhCDAB-
overexpressing cells exhibited the most improved butanol-
tolerance: a $3-fold improvement in growth compared to
wild-type cells at 1% (vol/vol) butanol. However, the glpC-
overexpressing cells did not show any butanol-tolerance,
even though it was reported that overexpression of glpC
contributes to an improvement of an organic solvent
tolerance of E. coli (Shimizu et al., 2005). In addition, co-
expression of sdhCDAB,flu,ybgD, and glpC in wild-type
cells led to more butanol-tolerance than the wild-type
cells overexpressing each gene individually, but much less
tolerance than the BT butanol-tolerant cells. Overexpression
of a single gene (or a few genes) did not produce a butanol-
tolerant phenotype that was as robust as the phenotype
induced by the BT ATF. It appears that the ability of ATFs of
eliciting simultaneous modification to expression levels of
many genes is more important for obtaining highly
improved butanol-tolerant phenotypes than modification
of expression levels of a few selected genes. We reason that
the change induced in the E. coli’s plasma membrane
through overexpression of SdhCDAB, Flu, and YbgD,
membrane proteins that are attached to, or associated with,
the cellular membrane, might provide structural stability to
the cell. This result is also consistent with a previous report
that overexpression of an outer membrane protein increases
an organic solvent tolerance of E. coli by reducing the influx
of an organic solvent (Abe et al., 2003).
In addition, we noticed that the BT ATF represses several
genes encoding iron-using proteins (Table II). This may lead
to an increase in the availability of intracellular iron. Iron is
an essential and beneficial element, acting as a cofactor for
many proteins where it plays important catalytic and/or
structural roles (Shiloach and Fass, 2005). Therefore, we
examined the effects of iron on the butanol-tolerance of
cells. When supplemented with 0.075 g/L of iron, which is
used for high cell density cultivation of E. coli (Shiloach and
Fass, 2005), the wild-type cells showed an improvement in
butanol-tolerance under the present butanol-stress condi-
tions (Fig. 3B). In particular, the wild-type cells showed $3-
fold improvement in growth in the presence of iron at 1.4%
(vol/vol) butanol (relative to its absence) (Fig. 3B).
However, the BT butanol-tolerant cells did not show any
Figure 4. Heat resistance of the butanol-tolerant cells (BT). Growth of cells was monitored after incubation for 2 h at 308C (no shock) or for 2 h at 558C (heat shock).
The triangles above each panel indicate 10-fold serial dilutions (1:1–1:1,000, left to right) of spotted cells. C, the wild-type E. coli transformed with a control plasmid. BT, the butanol-
tolerant cells expressing BT ATF. sdhCDAB,flu,ybgD, and glpC, cells overexpressing sdhCDAB ,flu,ybgD, and glpC, respectively. sþfþyþg, cells co-expressing sdhCDAB,
flu,ybgD, and glpC.
6Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2010
further increase in butanol-tolerance when grown in the
presence of iron, implying that the increased intracellular
iron availability by the BT ATF may be sufficient to cause
butanol-tolerance.
Taken together, it is likely that the highly improved
butanol-tolerant phenotype is caused by sophisticated
orchestration of the expression of many relevant genes.
Heat Resistance of Butanol-Tolerant Cells
Butanol increases the fluidity of the cellular membranes and
disrupts the function of embedded membrane proteins
(Piper, 1995). The toxic effects of butanol are similar to
those of heat shock, which also leads to increased membrane
fluidity (Sinensky, 1974). Therefore, we examined the heat
resistance of the selected BT butanol-tolerant cells (Fig. 4).
We also examined the effects of overexpression of sdhCDAB,
flu,ybgD, and glpC (aforementioned genes involved in
butanol-tolerance) on the heat resistance. After heat
treatment of the BT butanol-tolerant cells and sdhCDAB-,
flu-, ybgD-, or/and glpC-overexpressing cells at 558C for 2 h,
a condition that severely inhibits the growth of wild-type
E. coli, cells were plated on LB agar plates and incubated
overnight at 308C. The BT butanol-tolerant cells not only
survived but grew well even after the heat treatment (at
558C for 2 h), whereas the growth of wild-type cells was
completely inhibited under the same experimental condi-
tions. When the sdhCDAB,flu, or ybgD gene was
individually overexpressed in wild-type cells, the resulting
cells were more heat resistant than wild-type cells, but less so
than the BT butanol-tolerant cells. The glpC-overexpressing
cells, which did not show butanol-tolerance, were also more
heat resistant than wild-type cells, but much less so than the
BT butanol-tolerant and the sdhCDAB-, flu-, or ybgD-
overexpressing cells. In addition, the sdhCDAB,flu,ybgD,
and glpC co-expressing cells were nearly as heat resistant as
the BT butanol-tolerant cells. The wild-type, the BT
butanol-tolerant, and the sdhCDAB-, flu-, ybgD-, or/and
glpC-overexpressing cells showed similar growth patterns
when grown at 308C. This suggests that the butanol and heat
shock stresses of E. coli exhibit close similarity in their
responses and a functional overlap. The mechanism of the
resistance to butanol and heat shock remains to be examined
in detail. Nevertheless, it appears that the ATF elicits
simultaneous modification to expression levels of many
genes and changes an entire gene expression network in cells,
which leads to simultaneous resistance to butanol and heat
shock. Further identification and characterization of genes
regulated by the BT ATF will likely enhance our under-
standing of the complex phenotypes of butanol and heat
shock responses and those common control mechanisms
involved. Furthermore, butanol-tolerant E. coli capable of
growing at high temperature is very important for effective
butanol production, as butanol production at high
temperature increases the productivity and allows for facile
recovery of butanol, thereby making it possible to achieve
high-yield butanol production (Gong et al., 1999).
This work was supported by grants from the 21C Frontier Program of
Microbial Genomics and Applications (MG08-0204-1-0) and the
Research Program of New Drug Target Discovery (M10748222314-
08N4800-31410) from the Ministry of Education, Science and Tech-
nology of Korea, the Basic Program of the Korea Science and
Engineering Foundation (R01-2008-000-20559-0) and the Korea-
Australia Collaborative Research Project on the Development of
Sucrose-Based Bioprocess Platform (N02071165) from the Korean
Ministry of Knowledge Economy.
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