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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

([email protected])

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

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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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),


http://dx.doi.org/10.1016/j.copbio.2011.01.011

mailto:[email protected]

mailto:[email protected]


http://www.bp.com/

2 Tissue, cell and pathway engineering


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-



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




Metabolic engineering of Clostridium acetobutylicum Lutke-Eversloh and Bahl 3


Please cite this article in press as: Lutke-Eversloh T, Bahl H. Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol (2011),

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

Current Opinion in Biotechnology

www.sciencedirect.com Current Opinion in Biotechnology 2011, 22:1–14




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


( 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).


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


: 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





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



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.






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



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






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.



[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






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



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�].






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



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






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

1.�

Jones DT, Woods DR: Acetone-butanol fermentation revisited.Microbiol Rev 1986, 50:484-524.



Although 25 years old, this review nicely outlines the details on thephysiology and metabolic stoichiometry of Clostridium acetobutylicum.By far the most cited review on ABE fermentation until today.

2. Durre P: Biobutanol: an attractive biofuel. Biotechnol J 2007,2:1525-1534.

3. Durre P: Fermentative butanol production: bulk chemical andbiofuel. Ann NY Acad Sci 2008, 1125:353-362.

4.�

Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS:Fermentative butanol production by clostridia. BiotechnolBioeng 2008, 101:209-228.

Detailed review on various aspects of the ABE fermentation in general,with an emphasis on the urgent need of systems biology approaches.

5. Gheshlaghi R, Scharer JM, Moo-Young M, Chou CP: Metabolicpathways of clostridia for producing butanol. Biotechnol Adv2009, 27:764-781.

6. Zheng YN, Li LZ, Xian M, Ma YJ, Yang JM, Xu X, He DZ: Problemswith the microbial production of butanol. J Ind MicrobiolBiotechnol 2009, 36:1127-1138.

7. Huang H, Liu H, Gan YR: Genetic modification of criticalenzymes and involved genes in butanol biosynthesis frombiomass. Biotechnol Adv 2010, 28:651-657.

8. Zverlov VV, Berezina O, Velikodvorskaya GA, Schwarz WH:Bacterial acetone and butanol production by industrialfermentation in the Soviet Union: use of hydrolyzedagricultural waste for biorefinery. Appl Microbiol Biotechnol2006, 71:587-597.

9. Ni Y, Sun Z: Recent progress on industrial fermentativeproduction of acetone–butanol–ethanol by Clostridiumacetobutylicum in China. Appl Microbiol Biotechnol 2009,83:415-423.

10.�

Kharkwal S, Karimi IA, Chang MW, Lee DY: Strain improvementand process development for biobutanol production. Rec PatBiotechnol 2009, 3:202-210.

Interesting review with an exclusive focus on recent patent applications.

11. Liu S, Qureshi N: How microbes tolerate ethanol and butanol.Nat Biotechnol 2009, 26:117-121.

12.�

Ezeji T, Milne C, Price ND, Blaschek HP: Achievements andperspectives to overcome the poor solvent resistance inacetone and butanol-producing microorganisms. ApplMicrobiol Biotechnol 2010, 85:1697-1712.

A recent review focussed on butanol toxicity issues. Besides an updateon the authors’ expertise on fermentation process engineering, metabolicengineering efforts on innate and heterologous butanol producers toimprove solvent resistance are compiled.

13. Nicolaou SA, Gaida SM, Papoutsakis ET: A comparativeview of metabolite and substrate stress and tolerance inmicrobial bioprocessing: From biofuels and chemicals, tobiocatalysis and bioremediation. Metab Eng 2010,12:307-331.

14. Ezeji TC, Qureshi N, Blaschek HP: Butanol fermentationresearch: upstream and downstream manipulations. ChemRec 2004, 4:305-314.

15. Ezeji TC, Qureshi N, Blaschek HP: Bioproduction of butanol frombiomass: from genes to bioreactors. Curr Opin Biotechnol 2007,18:220-227.

16. Qureshi N, Ezeji TC: Butanol, ‘a superior biofuel’ productionfrom agricultural residues (renewable biomass): Recentprogress in technology. Biofuels Bioprod Biorefin 2008,2:319-330.

17. Durre P, Hollergschwandner C: Initiation of endospore formationin Clostridium acetobutylicum. Anaerobe 2004, 10:69-74.

18.�

Paredes CJ, Alsaker KV, Papoutsakis ET: A comparativegenomic view of clostridial sporulation and physiology. NatRev Microbiol 2005, 3:969-978.

Thus far a unique review on clostridial sporulation and related genomics.

19.�

Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbialcellulose utilization: fundamentals and biotechnology.Microbiol Mol Biol Rev 2002, 66:506-577.

A very detailed review on the mechanisms of cellulose degradation.






20. Lynd LR, van Zyl WH, McBride JE, Laser M: Consolidatedbioprocessing of cellulosic biomass: an update. Curr OpinBiotechnol 2005, 16:577-583.

21. Demain AL, Newcomb M, Wu JHD: Cellulase, clostridia, andethanol. Microbiol Mol Biol Rev 2005, 69:124-154.

22.�

Fontes CM, Gilbert HJ: Cellulosomes: highly efficientnanomachines designed to deconstruct plant cell wallcomplex carbohydrates. Annu Rev Biochem 2010, 79:655-681.

An appropriate update of [19�].

23.�

Alper H, Stephanopoulos G: Engineering for biofuels: exploitinginnate microbial capacity or importing biosynthetic potential?Nat Rev Microbiol 2009, 7:715-723.

An expert’s view on fundamental consolidated bioprocessing issues.

24. Weber C, Farwick A, Benisch F, Brat D, Dietz H, Subtil T, Boles E:Trends and challenges in the microbial production oflignocellulosic bioalcohol fuels. Appl Microbiol Biotechnol 2010,87:1303-1315.

25.��

Papoutsakis ET: Engineering solventogenic clostridia. CurrOpin Biotechnol 2008, 19:420-429.

Excellent state-of-the-art review: it concisely summarizes major strate-gies for sustainable metabolic engineering of clostridial butanol produc-tion.

26. Mitchell WJ, Albasheri KA, Yazdanian M: Factors affectingutilization of carbohydrates by clostridia. FEMS Microbiol Rev1995, 17:317-329.

27. Servinsky MD, Kiel JT, Dupuy NF, Sund CJ: Transcriptionalanalysis of differential carbohydrate utilization by Clostridiumacetobutylicum. Microbiology 2010, 156:3478-3491.

28. Grimmler C, Held C, Liebl W, Ehrenreich A: Transcriptionalanalysis of catabolite repression in Clostridiumacetobutylicum growing on mixtures of D-glucose and D-xylose. J Biotechnol 2010, 150:315-323.

29. Ren C, Gu Y, Hu S, Wu Y, Wang P, Yang Y, Yang C, Yang S,Jiang W: Identification and inactivation of pleiotropic regulatorCcpA to eliminate glucose repression of xylose utilization inClostridium acetobutylicum. Metab Eng 2010, 12:446-454.

30. Bahl H, Gottschalk G: Parameters affecting solvent productionby Clostridium acetobutylicum in continuous culture.Biotechnol Bioeng Symp 1984, 14:215-223.

31. Bahl H, Gottwald M, Kuhn A, Rale, Andersch W, Gottschalk G:Nutritional factors affecting the ratio of solvents produced byClostridium acetobutylicum. Appl Environ Microbiol 1986,52:169-172.

32. Durre P: Formation of solvents in clostridia. In Handbook onClostridia. Edited by Durre P. Boca Raton, FL: CRC Press; 2005:671-693.

33. Chen JS: Alcohol dehydrogenase: multiplicity and relatednessin the solvent-producing clostridia. FEMS Microbiol Rev 1995,17:263-273.

34.�

Fontaine L, Meynial-Salles I, Girbal L, Yang X, Croux C,Soucaille P: Molecular characterization and transcriptionalanalysis of adhE2, the gene encoding the NADH-dependentaldehyde/alcohol dehydrogenase responsible for butanolproduction in alcohologenic cultures of Clostridiumacetobutylicum ATCC 824. J Bacteriol 2002,184:821-830.

This study first provided experimental evidence on the function of thebifunctional aldehyde/alcohol dehydrogenase 2 (AdhE2, CAP0035) incultures of C. acetobutyliucm with clearly reduced redox potential, theso-called alcohologenic cultures.

35. Grimmler C, Janssen H, Krauße D, Fischer RJ, Bahl H, Durre P,Liebl W, Ehrenreich A: Genome-wide gene expression analysisof the switch between acidogenesis and solventogenesis incontinuous cultures of Clostridium acetobutylicum. J MolMicrobiol Biotechnol 2011, 20:1-15.

36. Jones DT, van der Weshuizen A, Long S, Allcock ER, Reid SJ,Woods DR: Solvent production and morphological changes inClostridium acetobutylicum. Appl Environ Microbiol 1982,43:1434-1439.



37. Zhao Y, Tomas CA, Rudolph FB, Papoutsakis ET, Bennett GN:Intracellular butyryl phosphate and acetyl phosphateconcentrations in Clostridium acetobutylicum and theirimplications for solvent formation. Appl Environ Microbiol 2005,71:530-537.

38.�

Jones SW, Paredes CJ, Tracy B, Cheng N, Sillers R, Senger RS,Papoutsakis ET: The transcriptional program underlying thephysiology of clostridial sporulation. Genome Biol 2008,9:R114.

The most detailed transcriptome analyses on C. acetobutylicum batchcultures thus far, focussing on life cycle-related mechanisms with parti-cular emphasis on the molecular sporulation events.

39. Tracy BP, Jones SW, Papoutsakis ET: Inactivation of sE and sGin Clostridium acetobutylicum illuminates their roles inclostridial-cell form biogenesis, granulose synthesis,solventogenesis, and spore morphogenesis. J Bacteriol 2011doi: 10.1128/JB.01380-10.

40. Grupe H, Gottschalk G: Physiological events in Clostridiumacetobutylicum during the shift from acidogenesis tosolventogenesis in continous culture and presentationof a model for shift induction. Appl Environ Microbiol 1992,58:3896-3902.

41. Rao G, Ward PJ, Mutharasan R: Manipulation of end-productdistribution in strict anaerobes. Ann NY Acad Sci 1987,506:76-83.

42. Girbal L, Soucaille P: Regulation of Clostridium acetobutylicummetabolism as revealed by mixed-substrate steady-statecontinuous cultures: role of NADH/NAD ratio and ATP pool.J Bacteriol 1994, 176:6433-6438.

43. Girbal L, Croux C, Vasconcelos I, Soucaille P: Regulation ofmetabolic shifts in Clostridium acetobutylicum ATCC 824.FEMS Microbiol Rev 1995, 17:287-297.

44.�

Girbal L, Soucaille P: Regulation of solvent production inClostridium acetobutylicum. Trends Biotechnol 1998, 16:1-16.

This group did some pioneering work on the redox balance of C.acetobutylicum’s metabolism in the past which is summarized in thisbrief review. Unfortunately, it is the authors’ most recent journal review.

45. Nakayama S, Kosaka T, Hirakawa H, Matsuura K, Yoshino S,Furukawa K: Metabolic engineering for solvent productivity bydownregulation of the hydrogenase gene cluster hupCBA inClostridium saccharoperbutylacetonicum strain N1–4. ApplMicrobiol Biotechnol 2008, 78:483-493.

46. Schut GJ, Adams MW: The iron-hydrogenase of Thermotogamaritima utilizes ferredoxin and NADH synergistically: a newperspective on anaerobic hydrogen production. J Bacteriol2009, 191:4451-4457.

47. Hedderich R, Forzi L: Energy-converting [NiFe]-hydrogenases:more than just H2 activation. J Mol Microbiol Biotechnol 2005,10:92-104.

48.�

Calusinska M, Happe T, Joris B, Wilmotte A: The surprisingdiversity of clostridial hydrogenases: a comparative genomicperspective. Microbiology 2010, 156:1575-1588.

A nice review which comprehensively summarizes current knowledge onclostridial hydrogenases based on genomic data and recent literature.

49. Seedorf H, Fricke WF, Veith B, Bruggemann H, Liesegang H,Strittmatter A, Miethke M, Buckel W, Hinderberger J, Li F et al.: Thegenome of Clostridium kluyveri, a strict anaerobe with uniquemetabolic features. Proc Nat Acad Sci USA 2008, 105:2128-2133.

50.�

Li F, Hinderberger J, Seedorf H, Zhang J, Buckel W, Thauer RK:Coupled ferredoxin and crotonyl coenzyme A (CoA) reductionwith NADH catalyzed by the butyryl-CoA dehydrogenase/Etfcomplex from Clostridium kluyveri. J Bacteriol 2008, 190:843-850.

This and the following Ref. [51�] were important contributions for theunderstanding of the energetic coupling and electron bifurcation mechan-isms in C. kluyveri.

51.�

Wang S, Huang H, Moll J, Thauer RK: NADP+ reduction withreduced ferredoxin and NADP+ reduction with NADH arecoupled via an electron bifurcating enzyme complex inClostridium kluyveri. J Bacteriol 2010, 192:5115-5123.

See comment on [50�].



http://dx.doi.org/10.1128/JB.01380-10




52.��

Herrmann G, Jayamani E, Mai G, Buckel W: Energy conservationvia electron-transferring flavoprotein in anaerobic bacteria. JBacteriol 2008, 190:784-791.

Brilliant review connecting recent findings with biochemical knowledgeon anaerobic energy metabolism.

53. Biegel E, Schmidt S, Muller V: Genetic, immunological andbiochemical evidence for a Rnf complex in the acetogenAcetobacterium woodii. Environ Microbiol 2009, 11:1438-1443.

54.�

Biegel E, Muller V: Bacterial Na+-translocating ferredoxin:NAD+

oxidoreductase. Proc Natl Acad Sci USA 2010, 107:18138-18142.

This recent study provided detailed biochemical evidence for the ener-getic coupling of ferredoxin oxidation and sodium ion translocation inAcetobacterium woodii.

55.��

Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q,Gibson R, Lee HM, Dubois J, Qiu D, Hitti J et al.: Genomesequence and comparative analysis of the solvent-producingbacterium Clostridium acetobutylicum. J Bacteriol 2001,183:4823-4838.

The early publication of the C. acetobutylicum genome sequence pavedthe way for the current knowledge of this unique microorganism.

56. Tomas CA, Alsaker KV, Bonarius HP, Hendriksen WT, Yang H,Beamish JA, Paredes CJ, Papoutsakis ET: DNA array-basedtranscriptional analysis of asporogenous, nonsolventogenicClostridium acetobutylicum strains SKO1 and M5. J Bacteriol2003, 185:4539-4547.

57. Alsaker KV, Paredes CJ, Papoutsakis ET: Design, optimizationand validation of genomic DNA microarrays for examining theClostridium acetobutylicum transcriptome. BiotechnolBioprocess Eng 2005, 10:432-443.

58. Paredes CJ, Senger RS, Spath IS, Borden JR, Sillers R,Papoutsakis ET: A general framework for designing andvalidating oligomer-based DNA microarrays and itsapplication to Clostridium acetobutylicum. Appl EnvironMicrobiol 2007, 73:4631-4638.

59.�

Hillmann F, Doring C, Riebe O, Ehrenreich A, Fischer RJ, Bahl H:The role of PerR in O2-affected gene expression of Clostridiumacetobutylicum. J Bacteriol 2009, 191:6082-6093.

First global transcriptional view on the oxidative stress response of C.acetobutylicum and its aerotolerant PerR mutant.

60. Shi Z, Blaschek HP: Transcriptional analysis of Clostridiumbeijerinckii NCIMB 8052 and the hyper-butanol-producingmutant BA101 during the shift from acidogenesis tosolventogenesis. Appl Environ Microbiol 2008,74:7709-7714.

61. Hemme CL, Mouttaki H, Lee YJ, Zhang G, Goodwin L, Lucas S,Copeland A, Lapidus A, Glavina del Rio T, Tice H et al.:Sequencing of multiple clostridial genomes related tobiomass conversion and biofuels production. J Bacteriol 2010,192:6494-6496.

62.�

Schaffer S, Isci N, Zickner B, Durre P: Changes in proteinsynthesis and identification of proteins specifically inducedduring solventogenesis in Clostridium acetobutylicum.Electrophoresis 2002, 23:110-121.

First proteome analysis on the solventogenic physiology of C. acetobu-tylicum.

63. Sullivan L, Bennett GN: Proteome analysis and comparison ofClostridium acetobutylicum ATCC 824 and Spo0A strainvariants. J Ind Microbiol Biotechnol 2006, 33:298-308.

64. Alsaker KV, Spitzer TR, Papoutsakis ET: Transcriptional analysisof spo0A overexpression in Clostridium acetobutylicum andits effect on the cell’s response to butanol stress. J Bacteriol2004, 186:1959-1971.

65. Mao S, Luo Y, Zhang T, Li J, Bao G, Zhu Y, Chen Z, Zhang Y, Li Y,Ma Y: Proteome reference map and comparative proteomicanalysis between a wild type Clostridium acetobutylicum DSM1731 and its mutant with enhanced butanol tolerance andbutanol yield. J Proteome Res 2010, 9:3046-3061.

66. Tomas CA, Beamish J, Papoutsakis ET: Transcriptional analysisof butanol stress and tolerance in Clostridium acetobutylicum.J Bacteriol 2004, 186:2006-2018.



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.

Comprehensive proteome study of acetogenesis and solventogenesisproviding respective reference maps for C. acetobutylicum usingsteady-state cells. The data were accompanied by detailed transcriptomeanalyses and provide an excellent basis for future systems biologyapproaches due to the high degree of reproducibility of chemostat cultures.

68.�

Amador-Noguez D, Feng XJ, Fan J, Roquet N, Rabitz H,Rabinowitz JD: Systems-level metabolic flux profilingelucidates a complete, bifurcated TCA cycle in Clostridiumacetobutylicum. J Bacteriol 2010, 192:4452-4461.

Simultaneously to [69�], metabolomic studies provided important insightsinto the central carbon metabolism of C. acetobutylicum.

69.�

Crown SB, Indurthi DC, Ahn WS, Choi J, Papoutsakis ET,Antoniewicz MR: Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824:isotopomer analysis, in vitro activities and expressionanalysis. Biotechnol J 2010 doi: 10.1002/biot.201000282.

See comment on [68�].

70. Papoutsakis ET: Equations and calculations for fermentationsof butyric acid bacteria. Biotechnol Bioeng 1984, 26:174-187.

71. Desai RP, Nielsen LK, Papoutsakis ET: Stoichiometric modelingof Clostridium acetobutylicum fermentations with non-linearconstraints. J Biotechnol 1999, 71:191-205.

72. Shinto H, Tashiro Y, Yamashita M, Kobayashi G, Sekiguchi T,Hanai T, Kuriya Y, Okamoto M, Sonomoto K: Kinetic modelingand sensitivity analysis of acetone–butanol–ethanolproduction. J Biotechnol 2007, 131:45-56.

73. Shinto H, Tashiro Y, Kobayashi G, Sekiguchi T, Hanai T, Kuriya Y,Okamoto M, Sonomoto K: Kinetic study of substratedependency for higher butanol production in acetone–butanol–ethanol fermentation. Proc Biochem 2008, 43:1452-1461.

74. Lee J, Yun H, Feist AM, Palsson BØ, Lee SY: Genome-scalereconstruction and in silico analysis of the Clostridiumacetobutylicum ATCC 824 metabolic network. Appl MicrobiolBiotechnol 2008, 80:849-862.

75. Senger RS, Papoutsakis ET: Genome-scale model forClostridium acetobutylicum. Part I. Metabolic networkresolution and analysis. Biotechnol Bioeng 2008,101:1036-1052.

76. Senger RS, Papoutsakis ET: Genome-scale model forClostridium acetobutylicum. Part II. Development of specificproton flux states and numerically determined sub-systems.Biotechnol Bioeng 2008, 101:1053-1071.

77. Haus S, Jabbari S, Millat T, Janssen H, Fischer RJ, Bahl H, King JR,Wolkenhauer O: A systems biology approach to investigate theeffect of pH-induced gene regulation on solvent production byClostridium acetobutylicum in continuous culture. BMC SystBiol 2011, 5:10.

78.�

Senger RS: Biofuel production improvement with genome-scale models: the role of cell composition. Biotechnol J 2010,5:671-685.

This review concisely discusses the current computational models for C.acetobutylicum, understandable also for non-bioinformatic researchers.

79. Mermelstein LD, Papoutsakis ET: In vivo methylation inEscherichia coli by the Bacillus subtilis phage phi3TImethyltransferase to protect plasmids from restriction upontransformation of Clostridium acetobutylicum ATCC 824. ApplEnviron Microbiol 1993, 59:1077-1081.

80. Soucaille P, Figge G, Croux C: Process for chromosomalintegration and DNA sequence replacement in clostridia.International patent WO 2008/040387.

81. Dong H, Zhang Y, Dai Z, Li Y: Engineering Clostridium strain toaccept unmethylated DNA. PLoS One 2010, 5:e9038.

82.�

Heap JT, Pennington OJ, Cartman ST, Minton NP: A modularsystem for Clostridium shuttle plasmids. J Microbiol Methods2009, 78:79-85.



http://dx.doi.org/10.1002/biot.201000282




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.

83.��

Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP: TheClosTron: a universal gene knock-out system for the genusClostridium. J Microbiol Methods 2007, 70:452-464.

First publication on a reliable and reproducible method for targeted andstable gene inactivation in C. acetobutylicum and other clostridia.

84. Shao L, Hu S, Yang Y, Gu Y, Chen J, Yang Y, Jiang W, Yang S:Targeted gene disruption by use of a group II intron (targetron)vector in Clostridium acetobutylicum. Cell Res 2007,17:963-965.

85. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM,Scott JC, Minton NP: The ClosTron: mutagenesis in Clostridiumrefined and streamlined. J Microbiol Methods 2010,80:49-55.

86. Sillers R, Chow A, Tracy B, Papoutsakis ET: Metabolicengineering of the non-sporulating, non-solventogenicClostridium acetobutylicum strain M5 to produce butanolwithout acetone demonstrate the robustness of the acid-formation pathways and the importance of the electronbalance. Metab Eng 2008, 10:321-332.

87. Tracy BP, Papoutsakis ET: Methods and compositions forgenetically engineering clostridia species. International patentWO 2009/137778.

88. Heap JT, Minton NP: Methods. International patent WO 2009/101400.

89. Cartman ST, Minton NP: Method of double crossoverhomologous recombination in clostridia. International patentWO 2010/084349.

90. Desai RP, Papoutsakis ET: Antisense RNA strategies formetabolic engineering of Clostridium acetobutylicum. ApplEnviron Microbiol 1999, 65:936-945.

91. Green EM, Boynton ZL, Harris LM, Rudolph FB, Papoutsakis ET,Bennett GN: Genetic manipulation of the acid formationpathways by gene inactivation in Clostridium acetobutylicumATCC 824. Microbiology 1996, 142:2079-2086.

92. Tummala SB, Welker NE, Papoutsakis ET: Design of antisenseRNA constructs for downregulation of the acetone formationpathway of Clostridium acetobutylicum. J Bacteriol 2003,185:1923-1934.

93. Tummala SB, Junne SG, Papoutsakis ET: Antisense RNAdownregulation of coenzyme A transferase combined withalcohol-aldehyde dehydrogenase overexpression leads topredominantly alcohologenic Clostridium acetobutylicumfermentations. J Bacteriol 2003, 185:3644-3653.

94. Sillers R, Al-Hinai MA, Papoutsakis ET: Aldehyde-alcoholdehydrogenase and/or thiolase overexpression coupled withCoA transferase downregulation lead to higher alcohol titersand selectivity in Clostridium acetobutylicum fermentations.Biotechnol Bioeng 2008, 102:38-49.

95.��

Jiang Y, Xu C, Dong F, Yang Y, Jiang W, Yang S: Disruption of theacetoacetate decarboxylase gene in solvent-producingClostridium acetobutylicum increases the butanol ratio. MetabEng 2009, 11:284-291.

The first example of engineering the central fermentative metabolism of C.acetobutylicum using the Targetron technology.

96. Soucaille P: Process for the biological production of n-butanolat high yield. International patent WO 2008/052973.

97. Meinecke B, Bahl H, Gottschalk G: Selection of anasporogenous strain of Clostridium acetobutylicum incontinous culture under phosphate limitation. Appl EnvironMicrobiol 1984, 48:1064-1065.

98. Clark SW, Bennett GN, Rudolph FB: Isolation andcharacterization of mutants of Clostridium acetobutylicumATCC 824 deficient in acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (EC 2.8.3.9) and in othersolvent pathway Enzymes. Appl Environ Microbiol 1989,55:970-976.



99. Stim-Herndon KP, Nair RV, Papoutsakis ET, Bennett GN: Analysisof degenerate variants of Clostridium acetobutylicum ATCC824. Anaerobe 1996, 2:11-18.

100. Cornillot E, Nair RV, Papoutsakis ET, Soucaille P: The genes forbutanol and acetone formation in Clostridium acetobutylicumATCC 824 reside on a large plasmid whose loss leads todegeneration of the strain. J Bacteriol 1997, 179:5442-5447.

101. Nair RV, Papoutsakis ET: Expression of plasmid-encoded aad inClostridium acetobutylicum M5 restores vigorous butanolproduction. J Bacteriol 1994, 176:5843-5846.

102. Lee JY, Jang YS, Lee J, Papoutsakis ET, Lee SY: Metabolicengineering of Clostridium acetobutylicum M5 for highlyselective butanol production. Biotechnol J 2009,4:1432-1440.

103. Cai X, Bennett GN: Improving the Clostridium acetobutylicumbutanol fermentation by engineering the strain for co-production of riboflavin. J Ind Microbiol Biotechnol 2010 doi:10.1007/s10295-010-0875-6.

104. Bailey JE, Sburlati A, Hatzimanikatis V, Lee K, Renner WA, Tsai PS:Inverse metabolic engineering: a strategy for directed geneticengineering of useful phenotypes. Biotechnol Bioeng 2002,79:568-579.

105. Santos CN, Stephanopoulos G: Combinatorial engineering ofmicrobes for optimizing cellular phenotype. Curr Opin ChemBiol 2008, 12:168-176.

106. Hermann M, Fayolle F, Marchal R, Podvin L, Sebald M,Vandecasteele JP: Isolation and characterization of butanol-resistant mutants of Clostridium acetobutylicum. Appl EnvironMicrobiol 1985, 50:1238-1243.

107. Matta-el-Ammouri G, Janati-Idrissi R, Rambourg JM,Petitdemange H, Gay R: Acetone butanol fermentation by aClostridium acetobutylicum mutant with high solventproductivity. Biomass 1986, 10:109-119.

108. Qureshi N, Blaschek HP: Recent advances in ABE fermentation:hyper-butanol producing Clostridium beijerinckii BA101. J IndMicrobiol Biotechnol 2001, 27:287-291.

109. Durre P, Kuhn A, Gottschalk G: Treatment with allyl alcoholselects specifically for mutants of Clostridium acetobutylicumdefective in butanol synthesis. FEMS Microbiol Lett 1986,36:77-81.

110. Rogers P, Palosaari N: Clostridium acetobutylicum mutantsthat produce butyraldehyde and altered quantities of solvents.Appl Environ Microbiol 1987, 53:2761-2766.

111. Bertram J, Durre P: Conjugal transfer and expression ofstreptococcal transposons in Clostridium acetobutylicum.Arch Microbiol 1989, 151:551-557.

112. Bertram J, Kuhn A, Durre P: Tn916-induced mutants ofClostridium acetobutylicum defective in regulation of solventformation. Arch Microbiol 1990, 153:373-377.

113. Babb BL, Collett HJ, Reid SJ, Woods DR: Transposonmutagenesis of Clostridium acetobutylicum P262:isolation and characterization of solvent deficient andmetronidazole resistant mutants. FEMS Microbiol Lett 1993,114:343-348.

114�

.Borden JR, Papoutsakis ET: Dynamics of genomic-libraryenrichment and identification of solvent-tolerant genes inClostridium acetobutylicum. Appl Environ Microbiol 2007,73:3061-3068.

This unconventional approach was only the second combinatorial exam-ple for screening defined C. acetobutylicum populations to allow retracingthe phenotype-related genotype.

115. Borden JR, Jones SW, Indurthi D, Chen Y, Papoutsakis ET: Agenomic-library based discovery of a novel, possiblysynthetic, acid-tolerance mechanism in Clostridiumacetobutylicum involving non-coding RNAs and ribosomalRNA processing. Metab Eng 2010, 12:268-281.

116�

.Dietrich JA, McKee AE, Keasling JD: High-throughput metabolicengineering: advances in small-molecule screening andselection. Annu Rev Biochem 2010, 79:563-590.



http://dx.doi.org/10.1007/s10295-010-0875-6

http://dx.doi.org/10.1007/s10295-010-0875-6




A nicely written review on the necessity of developing suitable high-throughput screening techniques for combinatorial metabolic engineer-ing strategies: examples, limits and perspectives are provided.

117��

. Tracy BP, Gaida SM, Papoutsakis ET: Development andapplication of flow-cytometric techniques for analyzing andsorting endospore-forming clostridia. Appl Environ Microbiol2008, 74:7497-7506.

The most inventive recent publication on the development of a sophis-ticated high-throughput screening method for clostridia based on the lifecycle-associated morphological changes.

118�

.Tracy BP, Gaida SM, Papoutsakis ET: Flow cytometry forbacteria: enabling metabolic engineering, synthetic biologyand the elucidation of complex phenotypes. Curr OpinBiotechnol 2010, 21:85-99.

A concise survey on flow cytometry applications from a technical point ofview, opportunities and challenges for explorative metabolic engineeringapproaches are discussed.

119. Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H,Yamamoto S, Okino S, Suzuki N, Yukawa H: Expression ofClostridium acetobutylicum butanol synthetic genes inEscherichia coli. Appl Microbiol Biotechnol 2008, 77:1305-1316.

120. Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM,Brynildsen MP, Chou KJ, Hanai T, Liao JC: Metabolicengineering of Escherichia coli for 1-butanol production.Metab Eng 2008, 10:305-311.

121. Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A,Ouellet M, Keasling JD: Metabolic engineering ofSaccharomyces cerevisiae for the production of n-butanol.Microb Cell Fact 2008, 7:36.

122. Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KL:Engineering alternative butanol production platforms inheterologous bacteria. Metab Eng 2009, 11:262-273.

123. Knoshaug EP, Zhang M: Butanol tolerance in a selection ofmicroorganisms. Appl Biochem Biotechnol 2009, 153:13-20.

124. Li J, Zhao JB, Zhao M, Yang YL, Jiang WH, Yang S: Screening andcharacterization of butanol-tolerant microorganisms. LettAppl Microbiol 2010, 50:373-379.



125. Berezina OV, Zakharova NV, Brandt A, Yarotsky SV, Schwarz WH,Zverlov VV: Reconstructing the clostridial n-butanol metabolicpathway in Lactobacillus brevis. Appl Microbiol Biotechnol2010, 87:635-646.

126. Kopke M, Held C, Hujer S, Liesegang H, Wiezer A, Wollherr A,Ehrenreich A, Liebl W, Gottschalk G, Durre P: Clostridiumljungdahlii represents a microbial production platform basedon syngas. Proc Natl Acad Sci USA 2010, 107:13087-13092.

127. Fischer CR, Klein-Marcushamer D, Stephanopoulos G: Selectionand optimization of microbial hosts for biofuels production.Metab Eng 2008, 10:295-304.

128. Dellomonaco C, Rivera C, Campbell P, Gonzalez R: Engineeredrespiro-fermentative metabolism for the production ofbiofuels and biochemicals from fatty acid-rich feedstocks.Appl Environ Microbiol 2010, 76:5067-5078.

129. Ranganathan S, Maranas CD: Microbial 1-butanol production:identification of non-native production routes and in silicoengineering interventions. Biotechnol J 2010, 5:716-725.

130�

.Atsumi S, Hanai T, Liao JC: Non-fermentative pathways forsynthesis of branched-chain higher alcohols as biofuels.Nature 2008, 451:86-89.

Application and genetic engineering of a novel non-natural biosyntheticpathway for second-generation biofuel production in E. coli.

131. Atsumi S, Liao JC: Metabolic engineering for advanced biofuelproduction from Escherichia coli. Curr Opin Biotechnol 2008,19:414-419.

132. Ghim CM, Kim T, Mitchell RJ, Lee SK: Synthetic biology forbiofuels: Building designer microbes from the scratch.Biotechnol Bioproc Eng 2010, 15:11-21.

133. Dellomonaco C, Fava F, Gonzalez R: The path for nextgeneration biofuels: successes and challenges in the era ofsynthetic biology. Microb Cell Fact 2010, 9:3.

134. Clomburg JM, Gonzalez R: Biofuel production in Escherichiacoli: the role of metabolic engineering and synthetic biology.Appl Microbiol Biotechnol 2010, 86:419-434.




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