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Metabolic engineering of Clostridium acetobutylicum: recentadvances to improve butanol productionTina Lutke-Eversloh and Hubert Bahl
The biosynthesis of the solvents 1-butanol and acetone is
restricted to species of the genus Clostridium, a diverse group
of Gram-positive, endospore forming anaerobes comprising
toxin-producing strains as well as terrestrial non-pathogenic
species of biotechnological impact. Among solventogenic
clostridia, Clostridium acetobutylicum represents the model
organism and general but yet important genetic tools were
established only recently to investigate and understand the
complex life cycle-accompanied physiology and its regulatory
mechanisms. Since clostridial butanol production regained
much interest in the past few years, different metabolic
engineering approaches were conducted — although
promising and in part successful strategies were employed, the
major breakthrough to generate an optimum phenotype with
superior butanol titer, yield and productivity still remains to be
expected.
Address
Department of Microbiology, Institute of Biological Sciences, University
of Rostock, Albert Einstein-Str. 3, 18051 Rostock, Germany
Corresponding authors: Lutke-Eversloh, Tina
([email protected]) and Bahl, Hubert
Current Opinion in Biotechnology 2011, 22:1–14
This review comes from a themed issue on
Tissue, cell and pathway engineering
Edited by Uwe T. Bornscheuer and Ali Khademhosseini
0958-1669/$ – see front matter
# 2011 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2011.01.011
IntroductionThe clostridial acetone–butanol–ethanol (ABE) fermen-
tation represents one of the oldest industrial fermentation
processes known, ranking second in scale only to ethanol
fermentation by yeast. In the early 1920s, Chaim Weiz-
mann, who later became Israel’s first president, discov-
ered the anaerobic bacterium Clostridium acetobutylicumwhich naturally produces acetone, butanol and ethanol in
a ratio of 3:6:1. The initial production plants for the ABE
fermentation were developed because of the World War
I-dependent demand of acetone for the cordite manu-
facture, but butanol was only an unwanted byproduct.
However, butanol became a more important product after
the war. Nevertheless, industrial ABE fermentation
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declined rapidly after the 1950s as a result of the cheaper
petrochemical production of butanol [1�,2]. As shown in
Figure 1, research activities in academia and industry
steeply increased in the early 1980s as a response to the oil
crisis in the 1970s with approximately equal efforts in
technical aspects, that is fermentation and downstream
processing, and research on physiology and genetics of
solventogenic clostridia. In the context of today’s general
interests in biofuels, scientific publications on clostridial
research increased again in the past few years, probably
enforced by DuPont’s and British Petrol’s announcement
in 2006 to reconstitute the industrial-scale ABE fermen-
tation in the United Kingdom (URL: http://www.bp.com,
press release date: June 20, 2006).
As a consequence, various review articles were published
recently, summarizing general aspects of the ABE fer-
mentation [2,3,4�,5–7], focussing on production countries
[8,9], patent review [10�], product toxicity and tolerance
[11,12�,13], as well as technical process development [14–16], respectively. Reviews on clostridial sporulation
[17,18�], cellulolytic clostridia [19�,20,21,22�], and con-
solidated bioprocessing perspectives (e.g., [23�,24]) are
also available.
The intention of this review paper is to specifically sum
up the development of metabolic engineering tools and
strategies for C. acetobutylicum to improve the innate
butanol production. As an update of E. T. Papoutsaki’s
review of 2008 [25��], engineering approaches conducted
within the past few years are highlighted and important
physiological aspects of the fermentative metabolism are
discussed.
Central metabolic pathways and theirregulationThe fermentation of sugars by clostridia typically causes
three different growth phases: first, exponential growth
and formation of acids, second, transition to stationary
growth phase with reassimilation of acids and concomitant
formation of solvents, and third, formation of endospores.
C. acetobutylicum can utilize a variety of carbohydrates,
including pentoses, hexoses, oligosaccharides and polysac-
charides — an important benefit for converting lignocellu-
lose hydrolysates intobiofuels. Although cellulosome genes
are present and expressed, C. acetobutylicum is not capable of
using cellulose as a substrate. Recent global transcriptional
and mutant analyses provided new insights into carbo-
hydrate utilization and regulatory constraints such as the
well-known carbon catabolite repression [26–29].
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
Current Opinion in Biotechnology 2011, 22:1–14
2 Tissue, cell and pathway engineering
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Figure 1
80
100
20
40
60
Pu
blic
atio
ns
0
1950 1960 1970 1980 1990 2000 2010
YearCurrent Opinion in Biotechnology
Scientific publications on solventogenic clostridia since 1950. More than
4500 reference hits obtained from database searches (PubMed, Scopus
and ISI Web of Science; as of December 2010) were screened according
to their relation to basic and applied research on butanol fermentation
and solventogenic clostridia in general. A total of 1238 publications since
1950 were considered except for conference abstracts, university
reports and references lacking author or source information. The pie
charts on top show the ratio between publications on physiology and
genetics (white) and those covering topics of fermentation and
downstream processing (black) for each decade.
As summarized in Figure 2, glucose is catabolized to
pyruvate via the Embden–Meyerhof–Parnas pathway
and acetyl-CoA is primarily formed by the pyruvate:fer-
redoxin oxidoreductase. Under certain growth conditions,
such as pH values >5 and iron limitation, lactate can be
the major fermentation product [30,31]. Acetate is syn-
thesized via phosphotransacetylase and acetate kinase
reactions with the latter reaction providing ATP. For
the biosynthesis of butyrate, two molecules of acetyl-
CoA are condensed to acetoacetyl-CoA, followed by a
reduction to butyryl-CoA, which is then converted to
butyrate via phosphotransbutyrylase and butyrate kinase
reactions with ATP generation.
As a reaction to the significant decrease of the pH in the
culture, which may destroy the essential proton gradient
across the membrane, C. acetobutylicum switches its metab-
olism from acidogenesis to solventogenesis. Acetate and
butyrate are reassimilated to their corresponding CoA
derivatives catalyzed by the acetoacetyl-CoA:acyl-CoA
transferase, with acetoacetyl-CoA as the CoA donor.
Particularly when reducing equivalents are limiting, acet-
oacetate is decarboxylated to acetone in order to drive the
transferase reaction by acetoacetate removal [32]. Butyr-
aldehyde and butanol dehyrdogenase activities, which
can be provided by different dehydrogenases, convert
butyryl-CoA to butyraldehyde and finally to butanol, the
major fermentation product of C. acetobutylicum (Figure
2a). However, the role of the different alcohol dehydro-
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Current Opinion in Biotechnology 2011, 22:1–14
genases and their regulation still remains to be elucidated
[33,34�,35].
The biphasic metabolism of C. acetobutylicum is tightly
associated with different growth stages, that is exponen-
tially growing cells mainly produce acetic and butyric
acid, while acetone and butanol are formed by stationary
cells (Figure 2b). After entering the stationary phase, cells
start to synthesize granulose as intracellular storage com-
pound, and these ‘‘clostridial stage’’ cells can be micro-
scopically distinguished from vegetative cells [36].
However, the regulatory mechanisms of granulose for-
mation and re-utilization are not known. Subsequently,
the sporulation process is initiated and the granulose
granula presumably serve as energy and carbon source
for endospore formation [1�,36]. The resistant spores are
able to survive for a long period of time and germinate
under suitable environmental conditions.
The role of the well-known sporulation regulator Spo0A
of Gram-positive bacteria has been shown to be important
for the initiation of endospore and solvent formation in C.acetobutylicum, but in regard of the quite complex net-
works, more regulatory factors are very likely to be
involved [17,18�]. However, despite the fact that Spo0A
is a major regulator of solventogenesis, sporulation itself is
not required for butanol and acetone production — more-
over, sporulation is not desirable. According to the
importance of the phosphorylated form of Spo0A for
the initiation of spore formation and its connection to
solventogenesis, the metabolic intermediate butyrylpho-
sphate was also found to play a putative regulatory role.
Experiments including for example, the quantification of
intracellular acetylphosphate and butyrylphosphate
levels, exhibited the association of high butyrylphosphate
concentrations with the reutilization of carboxylic acids as
well as the initiation of solvent biosynthesis, indicating
that butyrylphosphate might be a regulatory molecule as
well, possibly acting as a phosphodonor for transcriptional
factors [37].
Considering modern methodologies, the group of E. T.
Papoutsakis started to provide comprehensive insights
into the life cycle of C. acetobutylicum by analyzing the
growth-associated stages on a global genomic and tran-
scriptomic as well as on a morphological level with a
particular focus on sigma factors putatively involved in
sporulation [18�,38�]. So far, these explorative approaches
guided the researchers to potential candidates, and inac-
tivation of the transcriptional regulators sE and sG exhib-
ited an opportunity to uncouple sporulation and
solventogensis in batch fermentation [39].
In conclusion, only little is known on the regulatory
circuits and molecular mechanisms for the transition
between acidogenesis and solventogenesis and the onset
of the sporulation process. Although many efforts and
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
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Metabolic engineering of Clostridium acetobutylicum Lutke-Eversloh and Bahl 3
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doi:10.1016/j.copbio.2011.01.011
Figure 2
(a)
(b)
Glucose
Lactate Pyruvate
NAD+
2 H+
2 ATP
ADP + Pi
ADP + Pi
ATP
ATP
6.4
6.4
-0.2
-0.2
-5.4
-5.4
-5.4 -4.53.8
-14.6
-4.5
-6.5
0.5
-0.2
5.3
3.8
-6.5
Acetyl-CoA Acetaldehyde Ethanol
Glyco
lysis
Acetate Acetoacetyl-CoA Acetoacetate
3-Hydroxybutyryl-CoA
Butyrate
Butyryl-CoAButyryl-P Butyraldehyde
Crotonyl-CoA
Acetone
Butanol
Acetyl-P
2 ADP + Pi
CoA
CoA
CoA
+ CoA
Ac-CoA/Bu-CoAAc/Bu
CoAAAc-CoA
AAc-CoA
AAc
AAc
Acetyl-CoA
Hyd
Hbd
Ptb AdhE AdhE
Crt
Bcd
CtfAB Adc
CtfAB
CtfAB
Ldh Pdc
Pfor
AdhE AdhE
Thl
PtaAck
Buk
Pi
Pi
Fdox
Fdred
Etfox
Etfred
CO2
H2O
CO2
CO2H2
2 NADH + H+
NADH + H+
NAD+
NADH + H+
NADH + H+
NADH + H+
2 NAD+
NAD+
NAD+ + CoA
NAD+
NADH+ H+
NAD(P)+
NAD(P)+
NAD(P)H+ H+
NAD(P)H+ H+
ACIDOGENESIS
SOLVENTOGENESIS
Forespores
Sporulation
DifferentiationSpore germination
Spore maturation
Growth
Vegetative cells
Spores Clostridial form
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4 Tissue, cell and pathway engineering
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Table 1
Stoichiometric reactions for C. acetobutylicum.
Reaction Substrate Products ATP yield
(1) 1 Glucose 2 Acetate + 2 CO2 + 4 H2 4
(2) 1 Glucose 1 Butyrate + 2 CO2 + 2 H2 3
(3) 1 Glucose 0.6 Acetate + 0.7 butyrate + 2 CO2 + 2.7 H2 3.3
(4) 1 Glucose 2 Lactate 2
(5) 1 Glucose 1 Acetate + 1 ethanol + 2 CO2 + 2 H2 3
(6) 1 Glucose 2 Ethanol + 2 CO2 2
(7) 1 Glucose 1 Acetone + 3 CO2 + 4 H2 2
(8) 1 Glucose 1 Butanol + 2 CO2 2
(9) 1 Glucose 0.5 Acetone + 0.5 butanol + 2.5 CO2 + 2 H2 2
(10) 1 Glucose 0.3 Acetone + 0.6 butanol + 0.2 ethanol + 2.3 CO2 + 1.2 H2 2
(11) 1 Glucose + 1 acetate 1 Acetone + 1 ethanol + 3 CO2 + 2 H2 2
(12) 1 Glucose + 1 acetate 1 Acetone + 0.5 butanol + 3 CO2 + 2 H2 2
(13) 1 Glucose + 1 butyrate 1 Acetone + 1 butanol + 3 CO2 + 2 H2 2
Both actual and theoretical fermentative reactions were calculated according to balanced reducing equivalents; water and protons were not
considered. Neither other substrates than glucose, acetate and butyrate, nor specific physiological conditions were taken into account. See text for
details.
some progress were made, we do not have a detailed
picture on the regulation of solvent production in C.acetobutylicum. In addition to Spo0A, other regulators must
be involved in the solventogenic shift which specifically
control solventogenic operons. But what are the inducing
signals, how do the regulators interact, and how are the
regulatory networks connected? The answers to these
questions will certainly facilitate straightforward meta-
bolic engineering of C. acetobutylicum.
Considerations on redox balance andstoichiometryRegarding the metabolic pathways (Figure 2), ATP is
predominantly generated during acidogenesis, whereas
high NAD(P)H levels were proposed to induce solven-
togenesis [40]. Table 1 lists several possible stoichio-
metric reactions of glucose to the different
fermentation products considering both carbon and redox
balances. In practice, however, C. acetobutylicum does
usually not follow only one of the simple routes. Instead,
multiple products are formed, best approximated by
reactions (3) for the acidogenic and (10) for the solvento-
genic phase, respectively (Table 1). Another important
issue is that solventogenesis can only pursue when glu-
cose is concomitantly metabolized, which makes it diffi-
cult to stoichiometrically sum up all fermentation
products [1�]. But in theory, homo-butanol fermentation
is possible as shown by reaction (8) (Table 1): two moles
of ATP can be provided by glycolysis and the reducing
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( Figure 2 Legend ) Acidogenesis and solventogenesis in C. acetobutylicum
The red letters show the enzymes involved in the fermentative pathways: Ld
hydrogenase; Pfor, pyruvate:ferredoxin oxidoreductase; Fd, ferredoxin; Pta,
dehyrogenase; CtfAB, acetoacetyl-CoA:acyl-CoA transferase; Adc, acetoac
dehydrogenase; Crt, crotonase, Bcd, butyryl-CoA dehydrogenase; Etf, elect
kinase. AAc, acetoacetate; AAc-CoA, acetoacetyl-CoA; Ac/Bu, acetate/buty
reduced. The blue numbers represent the standard Gibbs energy changes a
respective growth stages. The major (green/blue) and minor (grey) metabolic
schemes of (a).
Current Opinion in Biotechnology 2011, 22:1–14
equivalents can be regenerated in the butanol pathway,
provided that reduced ferredoxin transfers its electrons to
NAD(P)+ and no molecular hydrogen is formed.
Manipulation of the redox balance has been demon-
strated to push the metabolism of C. acetobutylicumtowards butanol formation. Provision of artificial electron
carriers such as methyl viologen or neutral red, increasing
the hydrogen partial pressure or gassing with carbon
monoxide led to high butanol and low acetone production
rates; similar results were obtained by cultivation under
iron-limiting conditions or using whey as a substrate [41–43,44�,31]. All these techniques were employed to reduce
hydrogenase activities, and the lack of molecular hydro-
gen formation resulted in an electron flow towards buta-
nol for the regeneration of the NAD(P)+ pool. The fact
that the ferredoxin/hydrogenase node plays an important
role for butanol production was also demonstrated for C.saccharoperbutylacetonicum strain N1–4 [45]: employing
antisense RNA against the hupCBA gene cluster, which
encodes a hydrogen-uptake hydrogenase, the butanol/
acetone ratio was decreased from 2.9 to 1.3, because
the increased hydrogen formation rate caused a 76%
decrease in butanol production [45]. Therefore, targeting
the hydrogen production of C. acetobutylicum might con-
stitute a promising metabolic engineering strategy. Inter-
estingly, novel energetic properties of some hydrogenase
representatives were discovered more recently. The tri-
meric bifurcating hydrogenase of Thermotoga maritima
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
: Metabolic pathways (a) and their relation to the life cycle stages (b). (a)
h, lactate dehydrogenase; Pdc, pyruvate decarboxylase; Hyd,
phosphotransacetylase; Ack, acetate kinase; AdhE, aldehyde/alcohol
etate decarboxylase; Thl, thiolase; Hbd, 3-hydroxybutyryl-CoA
ron transfer flavoprotein; Ptb, phosphotransbutyrylase, Buk, butyrate
rate; Ac-CoA/Bu-CoA, acetyl-CoA/butyryl-CoA; ox, oxidized; red,
ccording to [6]. (b) Acidogenesis and solventogenesis referring to the
fluxes are indicated in the miniature pathways, which represent simplified
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Metabolic engineering of Clostridium acetobutylicum Lutke-Eversloh and Bahl 5
COBIOT-856; NO. OF PAGES 14
utilizes reduced ferredoxin and NADH simultaneously,
but C. beijerinckii is the only solventogenic strain that
potentially encodes a homologous protein [46]. Another
exciting group of enzymes are the energy-converting
[NiFe] hydrogenases which resemble the well-studied
complex I and are also designated as ‘‘respiratory’’ hydro-
genases [47]. Although similar genes were found in a few
clostridial species, none are present in the C. acetobutyli-cum genome [48�].
Yet another scientific breakthrough regarding anaerobic
energy metabolism was achieved recently, solving the
hitherto unbalanced stoichiometry of the C. kluyveri etha-
nol/acetate fermentation [49]. Here, the exergonic reac-
tion from crotonyl-CoA to butyryl-CoA is energetically
coupled with the endergonic reduction of ferredoxin by
NADH, which is catalyzed by the cytosolic Bcd/Etf
(butyryl-CoA dehydrogenase/electron transfer flavopro-
tein) complex [50�]. Analogously, the NADH-dependent
and ferredoxin-dependent reduction of NADP+ is
mediated by the electron bifurcating NfnAB (NADH-
dependent reduced ferredoxin:NADP+ oxidoreductase)
complex, an important link for NADPH generation since
a transhydrogenase gene is absent [51�].
The energy of the flavin-based electron bifurcation in C.kluyveri is conserved by pumping protons out of the cell,
which allows additional ATP generation. Thus, the dogma
of the past decades that substrate level phosphorylation is
the only energy source in fermenting bacteria was shown to
be wrong [52��]. The major player in the electron transport
phosphorylation in C. kluyveri is the membrane-associated
Rnf (Rhodobacter nitrogen fixation) complex providing
ferredoxin:NAD+ oxidoreductase activity, which occurs
in a variety of microbes and was recently biochemically
characterized in Acetobacterium woodii [53,54�]. According
to the available genome sequences, C. pasteurianum and C.acetobutylicum are the only clostridia lacking Rnf-homolo-
gous genes. Hence, C. acetobutylicum harbors a cytosolic
Bcd/Etf complex to reduce crotonyl-CoA, but obviously,
this reaction is not coupled to a membrane-associated
electron transport mechanism. The reason for the absence
of an energy conserving step is not known. We speculate
that because of the severe membrane damaging properties
of acids and solvents, C. acetobutylicum abandoned this
option for a reduced susceptibility to its own fermentation
products. Another reason might be a faster and probably
much more flexible energy metabolism, because all necess-
ary enzymes are soluble and usually, those proteins which
are located in the membrane constitute the limiting step of
a metabolic pathway. However, the fact that a similar or
different energy conserving mechanism has not been
found in C. acetobutylicum does not strictly exclude its
existence.
The above-mentioned findings on energy conservation in
anaerobic bacteria are quite new and almost nothing is
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known on the genetic regulation of the energy and redox
status in solventogenic clostridia, but more interesting
results can be expected in the near future. Again, soph-
isticated metabolic engineering approaches will be based
on the identification of the molecular regulatory switches,
sensing and transferring redox signals to induce or prolong
butanol synthesis. Turning such information to account,
C. acetobutylicum might be outflanked to increase the
carbon flow towards the desired product without redox-
based regulatory constraints.
Analytical and engineering tools forC. acetobutylicumAfter publication of the genome sequence of C. acetobu-tylicum ATCC 824 [55��], several transcriptome analyses
related to various physiological aspects such as sporula-
tion, solventogenesis or butanol stress were conducted by
the laboratory of E. T. Papoutsakis (e.g., [56–58]). Among
these, the most comprehensive DNA microarray study on
C. acetobutylicum batch cultures was published in 2008
[38�], providing detailed analyses on all relevant not yet
assigned sigma factors putatively involved in the sporula-
tion process. The first report from a different laboratory
employing DNA microarray methods was published only
in 2009, revealing transcriptional details on detoxification
and redox balance mechanisms in C. acetobutylicum related
to oxygen stress [59�]. Since recently, global transcrip-
tional analyses can also be performed with the related
strain C. beijerinckii NCIMB 8052 [60], but other solven-
togenic clostridia are thus far not accessible to DNA
microarray analyses, albeit other genomes are expected
to be sequenced or are in progress, respectively [61].
Proteome analyses can be regarded as a further step
towards understanding the solventogenic physiology
using ‘omics’ applications. The first system level two-
dimensional protein gels from chemostat cells were
already published in 2002, and major proteins induced
at the onset of solventogenesis such as the acetoacetate
decarboxylase were identified [62�]. Four years later,
comparative mass spectroscopic analyses of cytosolic
proteins related to the sporulation master regulator Spo0A
were conducted and compared accordingly to previous
transcriptome data [63,64]. The latter approach was done
using protein extracts from C. acetobutylicum batch cul-
tures, similar to a recent proteomic study of C. acetobuty-licum strain DSM 1731 as compared to its mutant strain
Rh8 obtained earlier by chemical mutagenesis. The phe-
notype of increased butanol tolerance and yield was
reflected in the proteome data — although not entirely
matching previous transcriptome results on butanol stress
experiments [65,66].
The emphasis on the cultivation conditions for the above
mentioned ‘omics’ publications can be explained as fol-
lows: simple batch fermentations, even if reproducible,
differ in general from continuous chemostat cultures.
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
Current Opinion in Biotechnology 2011, 22:1–14
6 Tissue, cell and pathway engineering
COBIOT-856; NO. OF PAGES 14
Whereas a batch fermentation resembles the natural life
cycle of C. acetobutylicum, chemostat cultivation is an
excellent research tool to keep the cells at a steady state,
that is all parameters are perfectly constant and the native
metabolism can be pushed into a desired direction, such
as uncoupling solventogenesis from sporulation. Using
steady state C. acetobutylicum cells, distinct proteome
reference maps for acidogenesis and solventogenesis
were generated and accompanied by detailed transcrip-
tome data [67��]. Such reference maps provide a suitable
basis for detailed single parameter analyses with a mini-
mum degree of disturbance, for example, specific exogen-
ous factors such as nutrient supply or defined stress
stimuli can be examined as well as the alteration of
endogenous factors like an interesting mutant strain.
Continuing the development of more sophisticated
systems biology methods, metabolome analyses can
provide important information on metabolic pathways
and fluxes. As a simple general rule for system
level approaches, the quality of information increases
with proximity to the organism’s actual physiology
(i.e., gene < protein < metabolite < pathway) which is
usually associated with increasing experimental complex-
ity and difficulty. Interestingly, the first metabolome data
sets on the central carbon metabolism of C. acetobutylicumderived from isotope tracer experiments were published
only recently, exhibiting a complete tricarboxylic acid
(TCA) cycle with a reductive and oxidative branch
towards succinate [68�,69�].
A common goal of various ‘omics’ strategies is the
development of a computational model resembling
the metabolic pathways and fluxes. Stoichiometric cal-
culations for modeling the ABE fermentation were
already described in 1984 [70], and Desai et al. [71]
conducted metabolic flux analyses of the acid reassi-
milation in C. acetobutylicum prior its genome sequence
was available. The first computational model for kinetic
simulations of the ABE fermentation was published in
2007 for C. saccharoperbutylacetonicum N1–4 [72,73].
Regarding C. acetobutylicum, genome-scale models were
independently developed by different research groups
[74–77] and are discussed in a recent review [78�].
The systems biology approaches and in silico models
described above provide a useful tool to predict metabolic
engineering targets, and experimental validation of such
targets generally requires much more efforts and respect-
ive literature is still sparse. The major reason for this is the
difficult genetic accessibility of clostridia, which ham-
pered successful engineering of C. acetobutylicum and
other solventogenic strains for a long time. However, a
number of suitable molecular protocols were developed
in recent years, although the clostridial portfolio of
genetic methods displays only a minor fraction as com-
pared to the metabolic engineering toolbox available for
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other model organisms such as Escherichia coli or Sacchar-omyces cerevisiae.
Regarding plasmid-based gene overexpression in C. acet-obutylicum, an improvement of the tedious in vivo meth-
ylation protocol [79] can now be achieved by using host
strains lacking the very active restriction endonuclease
Cac824I [80,81]. Furthermore, the range of available
plasmids was extended by the modular pMTL80000
series providing different selection markers, a choice of
Gram-positive and Gram-negative replicons, a multiple
cloning site, tra genes for conjugal transfer and allows
simplified cloning in E. coli using blue/white screening.
Depending on the application, the different shuttle plas-
mid modules can be assembled individually and com-
bined with the gene(s) of interest [82�].
The same research group also developed a reliable
method for directed gene disruption by adapting the
Targetron system to clostridial species, a similar paper
was published by Shao et al. [83��,84]. Further modifi-
cations of the ClosTron system allow by now the con-
struction of multiple knock-out (KO) mutants via
flippase-mediated marker recycling [85], providing an
important platform for sustainable metabolic engineering
of clostridial butanol production. Probably because of
commercial licensing issues, other research groups devel-
oped different KO methods for C. acetobutylicum and the
laboratory of N. P. Minton also filed a patent for a
ClosTron-independent KO system [80,86–89]. Until
now, the most often described method in the literature
to specifically decrease gene expression in C. acetobuty-liucm was the application of antisense RNA to ‘knock-
down’ clostridial genes [90].
Rational metabolic engineering strategiesAs shown in Figure 1, the ABE fermentation regained
much interest after the oil crisis in the 1980s and many
studies on optimizing the cultivation conditions and
varying the feeding regime were published. Since clos-
tridia were genetically not accessible at that time, other
approaches were chosen to investigate the physiology and
regulation of butanol biosynthesis. A major improvement
in butanol production by C. acetobutylicum and other
solventogenic strains was achieved by lowering the redox
potential to promote butanol formation, for example, by
using glycerol as co-substrate or addition of methyl violo-
gen (for review see e.g., [43,44�]). Within the past 15
years, overcoming the burden of genetic inaccessibility
was successful and the number of metabolic engineering
approaches for C. acetobutylicum steadily increased; refer-
ences published prior 2008 can be found elsewhere
[4�,25��], whereas recent efforts are listed in Table 2.
The major principle of rationally designing and improv-
ing a microbial production strain is to increase the meta-
bolic flux towards the desired product, usually
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
www.sciencedirect.com
Metabolic engineering of Clostridium acetobutylicum Lutke-Eversloh and Bahl 7
COBIOT-856; NO. OF PAGES 14
Table 2
Metabolic engineering of C. acetobutylicum for improved butanol production since 2005.
Parental strain Strategy Phenotype
(compared to control)
Genotype/plasmid Reference
C. acetobutylicum
ATCC 824
Dcac1515Dupp
Disruption of butyrate, acetone,
lactate, and acetate pathways
No information Dbuk, DctfAB, Dldh Dpta/ack [96]
C. acetobutylicum
ATCC 824
Dcac1515Dupp
Disruption of butyrate, acetone,
lactate, and hydrogen pathways
No information Dbuk, DctfAB, Dldh DhydA [96]
C. acetobutylicum
ATCC 824
Based on a previously published
idea, promotors for enhanced
adhE1 overexpression and ctfA
downregulation were exchanged
178 mM (176) butanol,
61 mM (109) acetone,
305 mM (20) ethanol,
2 mM (37) butyrate,
85 mM (77) acetate;
earlier butanol production
pCASAAD
(pptb-adhE1-pthl-asRNA:ctfB)
[93,94]
C. acetobutylicum M5 Based on a previously published
idea, the native sol promotor was
exchanged by the ptb promotor
for adhE1 expression in the
solvent-negative M5 strain
92 mM (84) butanol,
20 mM (8) ethanol,
72 mM (99) butyrate,
159 mM (101) acetate;
no acetone
p94AAD3 (pptb-adhE1) [86,101]
C. acetobutylicum M5 Expression of the sol operon in
the solvent-negative M5 strain
154 mM (69) butanol,
20 mM (9) ethanol,
10 mM (none) acetone,
54 mM (85) butyrate,
227 mM (168) acetate
pIMP1E1AB (psol-adhE1-ctfAB) [101,102]
C. acetobutylicum
EA 2018
Disruption of acetone pathway 100 mM (184) butanol,
36 mM (59) ethanol,
4 mM (49) acetone,
3 mM (6) butyrate,
60 mM (8) acetate;
80% (71) butanol ratio
of total solvents
adc::Int(180/181) [95��]
C. acetobutylicum
ATCC 824
Co-production of riboflavin as
a high-value product
70 mg/l (none) riboflavin,
193 mM (191) butanol
pJpGN (pptb-ribGBAH) [103]
Tables summarizing previous approaches can be found in [4�,32].
accomplished by reduced byproduct formation and elimi-
nated enzymatic bottle necks. Depending on the type of
product, metabolic engineering typically becomes more
and more difficult with increasing complexity of the
physiology and decreasing knowledge on regulatory
mechanisms. Thus, engineering C. acetobutylicum to
improve butanol production represents in fact a very
difficult goal because of the branched fermentative path-
way and a severe lack of information regarding regulatory
circuits determining the organism’s life cycle. Targeting
the elimination of the acid forming pathways in C. acet-obutylicum, buk-negative and pta-negative mutants were
already described by Green et al. [91] and overexpression
of adhE1 (also referred to as aad) in different host strains
for increased alcohol production was investigated, too (for
review see [4�,32]). Regarding the acetone branch, C.acetobutylicum was successfully engineered for reduced
acetone formation employing antisense RNA against ctfBto improve the butanol:acetone ratio [92]. However, the
mutant strain also exhibited reduced butanol production,
and therefore, the ctfB ‘knock-down’ strain was combined
with adhE1 overexpression. Interestingly, this strain not
only restored the butanol levels but also showed high
ethanol titers of up to 200 mM [93]. Recently, Sillers et al.
Please cite this article in press as: Lutke-Eversloh T, Bahl H. Metabolic engineering of Clostridiumdoi:10.1016/j.copbio.2011.01.011
www.sciencedirect.com
[94] picked up this strategy again, but optimized gene
expression of the asRNA:ctfB construct using the adcpromotor and the ptb promotor for higher adhE1 expres-
sion led to a more distinct phenotype (Table 2). On the
basis of metabolic flux analyses, the authors further co-
overexpressed the thl gene with adhE1 in order to stimu-
late the butyrate/butanol (C4) pathway. Although acetate
production could be reduced, the overall phenotype
comprising elevated thl expression in C. acetobutylicumwas not improved [94].
The first example of a Targetron-based KO mutant of C.acetobutylicum defective in the central fermentative
metabolism was published by Jiang et al. [95��] who
disrupted the adc gene by insertion of the group II intron.
The adc KO mutant exhibited small amounts of acetone
because of non-enzymatic decarboxylation of acetoace-
tate, but also significantly lower butanol titers as com-
pared to the parental strain C. acetobutylicum EA2018. The
butanol production was further improved by buffering the
fermentation media with calcium carbonate and addition
of methyl viologen. The results of this study basically
confirmed the phenotype of the asRNA strategies pub-
lished by the group of E. T. Papoutsakis, that is reduced
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
Current Opinion in Biotechnology 2011, 22:1–14
8 Tissue, cell and pathway engineering
COBIOT-856; NO. OF PAGES 14
acetone synthesis also led to a reduced butanol pro-
duction, although the earlier approaches targeted the ctfBgene because ‘knock-down’ of the adc gene was not
successful [92].
Recently, a comprehensive trial to effectively decrease
byproduct formation in C. acetobutylicum was the gener-
ation of two multiple-KO mutants, one comprising
deleted buk, ctfAB, ldh and pta-ack genes, the other
exhibited KOs of the genes buk, ctfAB, ldh and hydA genes
[96]. However, except for the butyrate titer and total
alcohol and acetone yield values of the buk single-KO
mutant, no phenotypic information on the performance or
fitness of the engineered strains was provided in this
patent application.
Because the solventogenesis is naturally accompanied by
the sporulation process — which eventually ceases buta-
nol production — an asporogenous C. acetobutylicum strain
might constitute an excellent starting point for metabolic
engineering. Non-sporulating variants of C. acetobutylicumDSM1731 which were still capable of solvent production
have been selected from continuous cultures after several
weeks of operation [97]. More popular asporogenous
strains are degenerated variants which lost the megaplas-
mid pSOL1, such as C. acetobutylicum M5 and DG1 [98–100]. The best studied pSOL1-encoded genes are those
responsible for solvent formation, that is adhE1 and ctfABwhich form the tricistronic sol operon, and the adjacent
adc gene, but most of the other relevant functions of
pSOL1 genes remain to be elucidated [3]. Complemen-
tation of C. acetobutylicum M5 with the adhE1 gene
restored butanol production without acetone formation,
further improvement was achieved by exchanging the
native sol promotor by the strong constitutive ptb promo-
tor for adhE1 expression [86,101] (Table 2). It is note-
worthy at this point that the adhE1 gene (CAP0162) is not
the only gene responsible for aldehyde/alcohol dehydro-
genase function in C. acetobutylicum, as it was erroneously
indicated in some publications. Overexpression of adhE2(CAP0035) in the degenerated strain C. acetobutylicumDG1 also restored butanol production without acetone
formation [34�].
However, the major fermentation product of the engin-
eered pSOL1-free strains described above was acetate,
accompanied by high butyrate concentrations, and differ-
ent attempts including KOs of ack and buk as well as co-
overexpression of thl did not alter this phenotypic pattern
[86]. A further attempt to address the high acid accumu-
lation, Lee et al. [102] co-overexpressed the adhE1 and
ctfAB genes in C. acetobutylicum M5: although acetate still
remained the major fermentation product, butanol titers
were increased whereas acetone production constituted
only 20% of the wild-type level. The authors speculated
that the high acetate concentrations were because of the
strain’s compensation for ATP generation and that
Please cite this article in press as: Lutke-Eversloh T, Bahl H. Metabolic engineering of Clostridiumdoi:10.1016/j.copbio.2011.01.011
Current Opinion in Biotechnology 2011, 22:1–14
alternative acetate forming pathways might exist in C.acetobutylicum [102].
Lastly, a simple approach from the economic point of
view is the idea to combine a bulk product with a value-
added product. Cai and Bennett [103] realized this inven-
tive strategy by engineering C. acetobutylicum for riboflavin
(vitamin B2) production: homologous overexpression of
the ribGBAH genes did not exhibit any negative effect on
butanol production, but instead a high value compound
was co-produced [103].
Combinatorial metabolic engineeringstrategiesThus far, rational metabolic engineering of solventogenic
clostridia as compiled above revealed only limited suc-
cess. This might be attributed to the small portfolio of
genetic tools for this bacterial group and hence, the
eventual success for generating a superior butanol produ-
cing strain can be expected in the future due to the recent
development of suitable techniques. On the other hand,
the principle of systematic approaches might comprise a
general limitation because of multiple unknown factors
constituting a specific phenotype. Since native butanol
synthesis is exclusively performed by solventogenic clos-
tridia, the accompanied branched fermentative pathways
(Figure 2) have not evolved for the reason to be branched
and complex, but to provide a distinct advantage for the
organism. Therefore, it might be the better alternative to
look for a strain according to its overall performance, that
is selecting an improved strain because of its phenotypic
characteristics, ideally combined with a gain of knowl-
edge on the factors which specifically led to the pheno-
type of interest. This issue has been addressed to other
biotechnological microbes previously and is often
referred to as ‘inverse metabolic engineering’ [104,105].
As a prerequisite, combinatorial approaches strictly
depend on the availability of suitable screening methods
to select the respective phenotype.
In the broadest sense, the oldest and easiest screening
procedure is mimicking nature: selection by the cell’s
survival of certain environmental conditions. In fact, this
screening method has been the most successful so far for
isolating better butanol producing clostridial strains.
Employing random chemical mutagenesis and butanol
exposure, butanol tolerant strains of C. acetobutylicum were
selected and exhibited enhanced butanol production
(e.g., [106,107]). More recently, mutant Rh8 of C. acet-obutylicum DSM 1731 was obtained by genome shuffling
and selection in the presence of high butanol concen-
trations [65]. The well-studied mutant C. beijerinckiiBA101 was selected in the presence of 2-deoxyglucose
after chemical mutagenesis, revealing increased amylo-
lytic activity and enhanced butanol production [108].
Details on general issues of butanol toxicity and tolerance
were reviewed recently [11,12�].
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
www.sciencedirect.com
Metabolic engineering of Clostridium acetobutylicum Lutke-Eversloh and Bahl 9
COBIOT-856; NO. OF PAGES 14
Other screening methods of the pre-genome era of C.acetobutylicum were described aiming to select solvent-
negative mutants for classical biochemical and genetic
analyses. These approaches included the use of suicide
substrates such as allyl alcohol and bromobutyrate and
solvent-negative mutants were selected because of sur-
vival and a colorimetric alcohol assay was employed as
well [98,109,110]. Furthermore, the streptococcal trans-
poson Tn916 was used for C. acetobutylicum to isolate
mutants defective in solvent formation, the first example
of the employment of defined populations which allow to
trace back responsible genes [111–113].
The second example of screening defined C. acetobutyli-cum populations was published more recently by Borden
and Papoutsakis [114�]. Plasmid-based genomic libraries
were generated and homologously expressed in C. acet-obutylicum, which were subjected to butanol challenges to
enrich those plasmids which conferred enhanced butanol
tolerance. Among the sixteen enriched genes, overex-
pression of the CAC1869-encoded transcriptional regu-
lator was verified to enhance butanol tolerance, although
details on the mechanism and the physiological role
remain to be elucidated [114�]. The suitability of this
combinatorial overexpression strategy for disclosing inter-
esting candidate targets was confirmed by optimizing and
extending the approach: screening the genomic library for
butyrate tolerance led to the identification of non-coding
RNAs mediating improved carboxylic acid tolerance
[115].
Whereas in general the choice of appropriate populations
is only restricted to the particular scientific claims, the
availability of suitable screening procedures largely
depends on the phenotype of interest. Regarding butanol,
ethanol and other colorless small molecules, screening
and phenotype selection in a high-throughput manner
according to the product quantities can only be performed
indirectly, that is the product of interest must be visual-
ized physically or (bio-) chemically, such as using chemi-
cal derivatization or (bio-) indicators like fluorescence or
auxotrophy applications [116�]. Such quantitative screen-
ing techniques are not available yet for C. acetobutylicumand other solventogenic strains, but considering the
promising potential of explorative strategies, global
approaches for significant phenotype improvements can
certainly be expected in the future.
An innovative novel screening technique was described
by Tracy et al. [117��], who developed a flow cytometry
assay for analyzing cell types of C. acetobutylicum,
applicable for high-throughput screenings on a single cell
level with optional fluorescence-assisted cell sorting
(FACS) [117��,118�]. Interestingly, the authors observed
a stronger correlation between solvent production and the
vegetative cell type than the clostridial form type (Figure
2b). However, this finding requires further investigation
Please cite this article in press as: Lutke-Eversloh T, Bahl H. Metabolic engineering of Clostridiumdoi:10.1016/j.copbio.2011.01.011
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and validation, since the relation of sporulating cells to
butanol production has been demonstrated previously
(e.g., [36]). Nevertheless, the application of FACS to
solventogenic clostridia opens up new perspectives
regarding phenotype selection from various populations
according to life cycle-associated events.
Other microbial butanol producersBecause of the portfolio of available physiological and
bioinformatic data, as well as a broad range of genetic
tools, well-studied microorganisms such as E. coli provide
an excellent scientific platform for biofuel production.
Such heterologous approaches do not only allow detailed
analytical methods without native regulatory constraints,
many prospects for further genetic manipulation and
metabolic engineering are provided. However, such
laboratory strains often lack important traits, that is good
growth rates and prototrophy, as well as substrate and/or
product tolerance, which makes them less appropriate for
industrial applications. Therefore, the demand of strain
robustness for large-scale production often contradicts
with the benefits to specifically engineer the microbes
on the molecular level. C. acetobutylicum comprises both,
above-mentioned benefits and disadvantages: as demon-
strated by industrial ABE fermentation, C. acetobutylicumand other solventogenic clostridia have been proven to
constitute robust production strains. Moreover, they have
obviously evolved mechanisms to maintain viability in an
increasingly toxic environment, that is to survive inher-
ently toxic butanol concentrations of up to 2% until the
cells turn to endurable spores to escape the stressful
conditions. On the other hand, detailed analytical and
metabolic engineering methods are just about to be
established — still being far behind from other model
organisms, but highly promising for the near future.
Since solventogenic clostridia represent the focus of this
review, recent attempts for recombinant butanol pro-
duction are summarized only briefly. The clostridial
butanol pathway was reconstructed in E. coli [119,120],
S. cerevisiae [121], Bacillus subtilis and Pseudomonas putida[122]. Considering butanol tolerance features, lactic acid
bacteria might present better production hosts [123,124]
and respective genes of C. acetobutylicum were successfully
introduced into Lactobacillus brevis [125]. Another inter-
esting host is the homoacetogen C. ljungdahlii because of
its ability to utilize CO2/CO and H2 as substrates, and
traces of butanol were detected in recombinant cells
expressing the C. acetobutylicum butanol biosynthetic
pathway [126]. All of these heterologous butanol produ-
cers provide a scientific ‘proof of principle’, but butanol
production rates were significantly below 1 g/l and are
thus far not competitive to solventogenic clostridia, which
naturally produce butanol at concentrations of approxi-
mately 13 g/l (without technical product removal during
fermentation). However, further investigations, process
optimization, metabolic engineering and synthetic
acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),
Current Opinion in Biotechnology 2011, 22:1–14
10 Tissue, cell and pathway engineering
COBIOT-856; NO. OF PAGES 14
biology approaches can sustainably improve recombinant
butanol production in the future [127–129].
Finally, as an innovatively different approach, the 2-
ketoacid pathway for alternative biofuel production in
E. coli was developed by the laboratory of J. C. Liao to
synthesize various alcohols via a non-natural pathway
[130�,131]. Several interesting reviews on general issues
of recombinant biofuel production were published
recently (e.g., [23�,24,132–134]).
ConclusionsThe conversion of the classic ABE fermentation into a
single product, that is butanol, process is a prerequisite
for a successful industrial revival. C. acetobutylicum as well
as all other related bacteria harbors complex metabolic
pathways with several branching points which makes it
difficult to direct the carbon flow exclusively to butanol.
The achievements in the past few years with respect to
the development of genetic tools are very promising for
sustainable metabolic engineering strategies and the data
gathered by ‘omics’ technologies allowed deeper insights
into the physiology of C. acetobutylcum. However, a major
drawback is the lack of knowledge on how the metabolic
shift from acid to solvent production is regulated on the
molecular level, for example, what are the inducing
signals, which regulators are involved, how do they
interact, and how are the regulatory networks connected.
Therefore, which products in which quantities are pro-
duced by C. acetobutylicum is a question of not only which
genes are present, but also how the carbon and electron
flow is regulated to maintain the redox balance. The
regulation is governed by three major issues: first, a
balanced redox status is of crucial importance for the
anaerobe C. acetobutylicum; second, the energy yield must
be as efficient as possible; and third, the survival of the
self-poisoning fermentation products must be ensured.
Numerous experimental examples have shown that C.acetobutylicum can be obliged to alter its product spectrum
to favor butanol production, and implementing the
respective molecular mechanisms will most likely reveal
new perspectives for metabolic engineering approaches.
We are optimistic that a butanol-only clostridial strain
can be generated. Whether inactivation of byproduct
formation, improved strain robustness, abandonment
of sporulation, tuning global regulators, etc. or combi-
nations thereof will lead to superior butanol producing
phenotypes offer challenging questions for metabolic
engineers.
References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:
� of special interest�� of outstanding interest
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Current Opinion in Biotechnology 2011, 22:1–14
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51.�
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Please cite this article in press as: Lutke-Eversloh T, Bahl H. Metabolic engineering of Clostridiumdoi:10.1016/j.copbio.2011.01.011
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67.��
Janssen H, Doring C, Ehrenreich A, Voigt B, Hecker M, Bahl H,Fischer RJ: A proteomic and transcriptional view of acidogenicand solventogenic steady-state cells of Clostridiumacetobutylicum in a chemostat culture. Appl MicrobiolBiotechnol 2010, 87:2209-2226.
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68.�
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The authors established a very useful shuttle plasmid collection forsimplified gene cloning and expression in C. acetobutylicum and otherclostridia with a wide range of application-dependent opportunities.
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