COBIOT-856; NO. OF PAGES 14 Please cite this article in press as: Lu ¨ tke-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 Available online at www.sciencedirect.com Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production Tina Lu ¨ tke-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: Lu ¨ tke-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 Introduction The 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 acetobutylicum which 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 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 their regulation The 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 into biofuels. 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]. www.sciencedirect.com Current Opinion in Biotechnology 2011, 22:1–14
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COBIOT-856; NO. OF PAGES 14
Available online at www.sciencedirect.com
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
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
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
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.
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
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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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
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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.��
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