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Metabolic Engineering 13 (2011) 373–382

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Metabolic Engineering
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Metabolic engineering of Clostridium tyrobutyricum for n-butanol production

Mingrui Yu a, Yali Zhang a, I-Ching Tang b, Shang-Tian Yang a,n
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA
Bioprocessing Innovative Company, 4734 Bridle Path Court, Dublin, OH 43017, USA

a r t i c l e i n f o abstract

Article history: Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to
Received 6 January 2011 overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium
Received in revised form acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase
23 March 2011
(thl) promoter. Butanol titer of 1.1 g/L was obtained in C. tyrobutyricum overexpressing adhE2. The
Accepted 14 April 2011
effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on
Available online 22 April 2011
butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in
Keywords: ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout
Aldehyde/alcohol dehydrogenase mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux
distribution in C. tyrobutyricum. The highest butanol titer of 10.0 g/L and butanol yield of 27.0%
(w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing
Clostridium tyrobutyricum
Knockout adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0 g/L with
Metabolic engineering 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance,
with 4 80% and  60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that
C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass.
& 2011 Elsevier Inc. All rights reserved.

1. Introduction fermentation usually suffers from low butanol yield ( 20% w/

w), titer ( o15 g/L) and productivity ( o0.5 g/L h) because of
With concerns about greenhouse gas emission and uncertainty butanol toxicity and production of other byproducts including
about the supply of oil, renewable biofuels have gained increasing acetone, ethanol, acetate and butyrate (Lee et al., 2008). Extensive
attentions (Fortman et al., 2008). Bioethanol, the current major metabolic engineering studies of solventogenic Clostridia have
biofuel, is not an ideal replacement of gasoline because of its low been performed aiming at improving butanol yield, productivity
energy density, high water solubility and high vapor pressure. and tolerance (Lee et al., 2008; Nicolaou et al., 2010; Papoutsakis,
Compared to ethanol, butanol is more hydrophobic, has a more 2008). Although production of byproducts acetone, acetate and
similar energy content (27 MJ/L) to that of gasoline (32 MJ/L) and butyrate decreased by overexpressing aldehyde/alcohol dehydro-
can be transported in the existing pipeline infrastructure. Thus, genase (aad) and thiolase (thl) coupled with down-regulation of
butanol is considered as a better drop-in biofuel than ethanol. CoA transferase (coat, the first enzyme in the acetone formation
Butanol can be produced by anaerobic microorganisms such as pathway) with antisense RNA in C. acetobutylicum, butanol titer
Clostridium acetobutylicum and Clostridium beijerinckii in acetone– and yield were not improved; instead, ethanol concentration was
butanol–ethanol fermentation (ABE fermentation), which was largely increased (Sillers et al., 2009). Butanol to acetone ratio was
once the second largest industrial fermentation in the world enhanced by knocking out acetoacetate decarboxylase (adc) and
(Zheng et al., 2009). In a typical ABE fermentation, butyrate and altering electron flow with the addition of methyl viologen (Jiang
acetate are produced first; the culture then undergoes a metabolic et al., 2009). A non-solventogenic mutant strain C. acetobutylicum
shift and solvents (butanol, acetone and ethanol) are formed M5, which has lost pSOL1 megaplasmid, was used as a host to
(Gheshlaghi et al., 2009). Due to the complicated metabolic produce butanol without acetone by overexpressing aad gene
pathways involving acidogenesis and solventogenesis plus (Sillers et al., 2008). However, large amounts of acetate and
spore-forming life cycle, ABE fermentation is difficult to control butyrate were accumulated due to the inability of the strain M5
or manipulate (Zheng et al., 2009). Furthermore, ABE to re-assimilate acid products, and butanol titer never exceeded
that in wild-type C. acetobutylicum. Due to complex metabolic
regulation in C. acetobutylicum, to date little success on enhancing
Corresponding author. Fax: þ614 292 3769. butanol titer and yield has been made by metabolic engineering
E-mail address: (S.-T. Yang). (Lee et al., 2008).

1096-7176/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
374 M. Yu et al. / Metabolic Engineering 13 (2011) 373–382

The Clostridia’s butanol biosynthesis pathway has also been C. tyrobutyricum with high butyrate tolerance ( 450 g/L) (Zhu and
heterologously expressed for butanol production in various Yang, 2003) also showed a good butanol tolerance (see results in
industrial microbes, including Escherichia coli (Atsumi et al., Section 3.6). Therefore, C. tyrobutyricum with a simple, native
2008; Bond-Watts et al., 2011; Nielsen et al., 2009; Shen and (only acidogenesis) and favorable metabolic pathway toward
Liao, 2008), Saccharomyces cerevisiae (Steen et al., 2008), Pseudo- butyryl-CoA should be an interesting industrial host for butanol
monas putida (Nielsen et al., 2009), Bacillus subtilis (Nielsen et al., production.
2009), and Lactobacillus brevis (Berezina et al., 2010). Although In this paper, we report the engineering of C. tyrobutyricum to
these microbes have been found to possess several desirable overexpress heterologous adhE2 gene for n-butanol production
industrial attributes, including fast cell growth, well studied from glucose. The effects of different promoters and replicons
genetics, and moderate to high butanol tolerance (Fischer et al., used in the expression plasmids and ack and ptb knockouts in the
2008; Knoshaug and Zhang, 2009), their ability to produce host cells on butanol production were also investigated and are
n-butanol has been disappointing, achieving a butanol titer reported in this paper. To our knowledge, this study presents the
of o4.7 g/L (Bond-Watts et al., 2011), much lower than the highest n-butanol titer and yield from heterologous expression of
desirable level for industrial production. n-butanol pathway.
In this work, we studied the feasibility of engineering
C. tyrobutyricum, an anaerobic acidogen naturally producing 2. Materials and methods
butyrate and acetate as two major products from glucose and
xylose (Liu and Yang, 2006a), for butanol production. We have 2.1. Bacterial strains and culture media
previously constructed C. tyrobutyricum mutants with phospho-
transacetylase (pta) and acetate kinase (ack) knockout to enhance C. tyrobutyricum ATCC 25755 was the parental strain of
butyrate production with a high yield of 40.4 g/g glucose (Liu various mutant strains (see Table 1) developed in this work.
et al., 2006b, 2006c; Zhu et al., 2005). These mutants, after C. acetobutylicum ATCC 824 was used to extract adhE2 gene from
adaptation in a fibrous bed bioreactor, could produce more than its genome. E. coli DH5a (Invitrogen, Carlsbad, CA) was used in the
50 g/L butyrate with a high butyrate to acetate ratio of 46 (g/g) preparation of recombinant plasmids and E. coli CA434 (Williams
(Liu et al., 2006b; Liu and Yang, 2006a). The high butyrate titer et al., 1990) as the donor strain in conjugation described later.
and butyrate/acetate ratio suggested a favorable metabolic path- Unless otherwise noted, C. tyrobutyricum and C. acetobutylicum
way from glucose to butyryl-CoA, which could be converted to were grown anaerobically at 37 1C in Reinforced Clostridial Med-
butanol by introducing aldehyde/alcohol dehydrogenase (adhE2) ium (RCM; Difco, Detroit, MI). Colonies were maintained on RCM
into C. tyrobutyricum (see Fig. 1). Since no acetone forming genes (15 g/L agar) plates in the anaerobic chamber. These media were
are present in C. tyrobutyricum, butanol yield should be high as it supplemented with 40 mg/mL erythromycin or 30 mg/mL thiam-
would be the major solvent product. Unlike solventogenic clos- phenicol for transformant selection. E. coli was grown aerobically at
tridia such as C. acetobutylicum, C. tyrobutyricum is not a native 37 1C and 250 rpm in Luria–Bertani (LB) media supplemented with
solventogenic bacterium and thus its heterologous butanol chloramphenicol (30 mg/mL), ampicillin (100 mg/mL) or kanamycin
biosynthesis pathway is not associated with sporulation (50 mg/mL).
and cell autolysis, which greatly limit butanol production in
native solventogenic clostridia (Ezeji et al., 2010). Furthermore, 2.2. Plasmids construction

Several recombinant plasmids were constructed and used to

Glucose create various butanol-producing C. tyrobutyricum mutant strains

Table 1
Pyruvate H Strains and plasmids used in this study.
Fd H+ Strain/plasmid Relevant characteristics Reference/source
CO fnor
ack pta adhE2 adh C. tyrobutyricum ATCC 25755 ATCC
Acetate Acetyl~P Acetyl-CoA Acetylaldehyde Ethanol AckKO Ack knockout Liu et al. (2006b)
thl NADH NADH PtbKO Ptb knockout Zhang (2009)
Ct(pCAAD) ATCC 25755 with pCAAD This study
Ct(pSAD42) ATCC 25755 with pSAD42 This study
hbd Ct(pMTL007) ATCC 25755 with pMTL007 This study
ctf Ct(pMAD72) ATCC 25755 with pMAD72 This study
3-hydroxybutyryl-CoA AckKO(pMAD72) AckKO with pMAD72 This study
crt HO AckKO(pMAD22) AckKO with pMAD22 This study
PtbKO(pMAD72) PtbKO with pMAD72 This study
E. coli CA434 E. coli HB101 with plasmid Williams et al.
bcd & etfAB NADH R702 (1990)
buk ptb adhE2 bdh
Butyrate Butyryl~P Butyryl-CoA Butyraldehyde Butanol
pCAAD ColE1 ori; AmpR; EmR; pIM13 Nair et al. (1994)
Fig. 1. Metabolic pathway in Clostridium tyrobutyricum. The pathways for butanol ori; Paad::aad
and ethanol formation shown in dotted lines are absent in wild-type C. tyrobutyr- pSAD42 ColE1 ori; AmpR; EmR; pIM13 This study
icum and are introduced by overexpressing adhE2 gene. Key enzymes and genes in ori; Pptb::adhE2
the pathway: hydrogenase (hyda); pyruvate: ferredoxin oxidoreductase (pfor); pMTL82151 ColE1 ori; CmR; pBP1 ori; TarJ Heap et al. (2009)
ferredoxin NAD þ oxidoreductase (fnor); acetate kinase (ack); phosphotransacety- pMTL007 ColE1 ori; CmR; pCB102 ori; Heap et al. (2007)
lase (pta); thiolase (thl); beta-hydroxybutyryl-CoA dehydrogenase (hbd); croto- TarJ
nase (crt); butyryl-CoA dehydrogenase (bcd); electron transferring flavoprotein pMAD72 From pMTL007; Pthl::adhE2 This study
(etf); phosphotransbutyrylase (ptb); butyrate kinase (buk); CoA transferase (ctf); pMAD22 From pMTL82151; This study
alcohol dehydrogenase (adh); butanol dehydrogenase (bdh); aldehyde-alcohol Pthl::adhE2
dehydrogenase (adhE2).
M. Yu et al. / Metabolic Engineering 13 (2011) 373–382 375

(see Table 1 and Supplementary Materials). Plasmid pCAAD con- (50 g/L glucose, 3 g/L yeast extract, 1 g/L tryptone, 2 g/L K2HPO4, 2 g/L
taining aad gene and native aad promoter from C. acetobutylicum KH2PO4, 0.2 g/L MgSO4, 0.01 g/L MnSO4, 0.01 g/L FeSO4, 0.01 g/L NaCl)
ATCC 824 was obtained from Prof. G.N. Bennett of Rice University supplemented with 40 mg/mL erythromycin or 25 mg/mL thiamphe-
(Nair et al., 1994). Plasmids pSAD42 and pMAD72 were created by nicol as required. Each bottle containing 40 mL of the medium was
cloning adhE2 into plasmids pSOS94 (Genebank no. AY187685) and inoculated with 0.4 mL of cells from an overnight culture in RCM. The
pMTL007 (Genebank no. EF525477), respectively. The construction fermentation was carried out at 37 1C and the medium pH was
of these recombinant plasmids is briefly described below. maintained between 5.0 and 6.5 by adding NaOH solution twice a
The adhE2 gene (Genebank accession no. AF321779) was PCR- day. Samples were taken at regular intervals to monitor cell growth,
amplified from C. acetobutylicum ATCC 824 genomic DNA (see substrate (glucose) consumption and production of butanol, ethanol,
Supplementary Materials for primer sequences). The PCR product acetic acid and butyric acid during the fermentation.
was purified and digested with BamHI and SfoI, and then ligated
into plasmid pSOS94 digested with the same restriction enzymes
2.5. AdhE2 enzyme activity assays
to generate recombinant pSAD42. To construct recombinant
pMAD72, adhE2 was PCR-amplified from pSAD42, and thl promo-
The activities of butyraldehyde dehydrogenase and butanol
ter (Genebank accession no. HM989902) was PCR-amplified from
dehydrogenase of adhE2 were measured by monitoring NADH
C. tyrobutyricum ATCC 25755 genomic DNA. The PCR product of
consumption at 365 nm according to the method described before
thl promoter (Pthl) was ligated into pGEM-T vector (Promega,
(Durre et al., 1987) with some modifications. Cells were collected
Madison, WI) to generate pGEM-T-Pthl. Plasmid pGEM-T-Pthl and
from 100 mL fermentation broth by centrifugation at 10,000g for
adhE2 PCR product were digested with BamHI and SacII and
10 min using tightly sealed centrifuge tubes purged by nitrogen
ligated to generate pGEM-T-PthladhE2. PthladhE2, from pGEM-
gas. The cell pellet was washed with Tris-HCl buffer (0.1 M, pH
T-PthladhE2 after treating with HindIII and SacII, was ligated into
7.5) once and resuspended in 5 mL the same Tris-HCl buffer. Lysis
plasmids pMTL007 and pMTL82151, after HindIII and SacII diges-
was carried out using a French press with one passage at 77 MPa.
tion, to generate pMAD72 and pMAD22, respectively. All recom-
Supernatant was collected by centrifugation at 15,000g for 10 min
binant plasmids were transformed into E. coli DH5a and purified
and used for enzyme activity assay. Enzyme activity was calcu-
plasmids were confirmed by DNA sequencing.
lated on the basis of a molar NADH extinction coefficient of
3.4 cm  1 mM  1. One unit of enzyme activity was defined as the
2.3. Transformation
amount of enzyme which converts 1 mmol NADH per minute
under the reaction conditions. Protein concentration in cell
Unless otherwise noted, all transformation procedures were
extract was determined using the Bio-Rad protein assay kit with
performed in an anaerobic chamber. Plasmids of pCAAD and
bovine serum albumin as standard.
pSAD42 were transformed into C. tyrobutyricum by electropora-
tion following the previously described method (Zhu et al., 2005).
Two mutants, Ct(pCAAD) and Ct(pSAD42), were obtained by 2.6. Butanol tolerance
transforming ATCC 25755 with pCAAD or pSAD42, respectively.
C. tyrobutyricum was also transformed with pMAD72 via con- To evaluate the butanol tolerance of various C. tyrobutyricum
jugation following previously described procedures with some mutants and C. beijerinckii 55025, cells were cultured in 50 mL
modifications (Purdy et al., 2002). The donor strain E. coli CA434 centrifuge tubes, with cap tightly sealed, in an anaerobic cham-
(E. coli HB101 with IncPb conjugative plasmid R702) was first ber. Each tube containing 5 mL RCM medium and butanol at a
transformed with the recombinant plasmid to be mobilized by concentration of up to 2% (v/v) was inoculated with 0.2 mL of cells
electroporation, and the transformed cells were collected by cen- from an overnight culture in RCM. Cell growth was monitored by
trifugation after culturing in the LB medium containing 30 mg/mL measuring the optical density at 600 nm (OD600) of the culture
chloramphenicol at 37 1C and 250 rpm overnight to reach OD600 broth. The specific growth rates at various initial butanol con-
1.5–2.0. About 3 mL of the donor E. coli cells were centrifuged and centrations were estimated from OD600 data.
then washed once in 1 mL of sterile phosphate-buffered saline (PBS)
and resuspended into 0.4 mL of the recipient C. tyrobutyricum cells,
2.7. Analytical methods
which were grown overnight to OD600 2.0–3.0 in the RCM medium
at 37 1C. The cell mixture was then pipetted onto well-dried RCM
Cell density was analyzed by measuring the optical density of
agar plate and incubated at 37 1C for 24 h for mating. One milliliter
cell suspension at 600 nm using a spectrophotometer (Shimadzu,
of the RCM medium was then applied to each conjugation plate
Columbia, MD, UV-16-1). The glucose concentration was deter-
to harvest cells, which were then re-plated on RCM plates with
mined with YSI 2700 Select Biochemistry Analyzer (Yellow
25 mg/mL thiamphenicol (selection for plasmid uptake) and 200 mg/
Springs, OH). Butanol, ethanol, acetic acid and butyric acid were
mL D-cycloserine (counter selection for E. coli). Plates were incubated
analyzed with a gas chromatograph (GC) (GC-2014 Shimadzu,
for 48–96 h or until colonies were apparent. Three mutants,
Columbia, MD) equipped with a flame ionization detector (FID)
Ct(pMAD72), AckKO(pMAD72), and PtbKO(pMAD72), were obtained
and a 30.0 m fused silica column (Stabilwax-DA, 0.25 mm film
by transforming pMAD72 into C. tyrobutyricum ATCC 25755, AckKO
thickness and 0.25 mm ID, Restek, Bellefonte, PA). The GC was
with ack knockout (Liu et al., 2006b) and PtbKO with ptb knockout
operated at an injection temperature of 200 1C with 1 mL of
(Zhang, 2009), respectively. AckKO was also transformed with
sample injected with an auto injector (AOC-20i, Shimadzu). The
pMAD22 to obtain AckKO(pMAD22). In addition, C. tyrobutyricum
column temperature was initially held at 80 1C for 3 min, then
ATCC 25755 was also transformed with pMTL007 (without adhE2
increased at a constant rate of 30 1C per min to 150 1C, and held at
gene) to obtain the strain Ct(pMTL007), which was used as a control
150 1C for 3.7 min.
in the fermentation kinetics study described below.

2.4. Fermentation kinetics 2.8. Statistical analysis

Unless otherwise noted, batch fermentations with C. tyrobutyricum Unless otherwise noted, all experiments were triplicated, and
mutants were carried out in serum bottles containing P2 medium the average values with standard errors are reported. Student’s
376 M. Yu et al. / Metabolic Engineering 13 (2011) 373–382

t-test analysis of fermentation data were performed using JMP shown). Furthermore, these mutants were not stable and could
with p ¼0.05 as the threshold for significant difference. not be maintained in liquid cultures, suggesting poor plasmid
stability in the host. It is thus clear that pCAAD and pSAD42 were
inefficient for high-level butanol production in C. tyrobutyricum.
3. Results and discussion
3.2. Butanol production by mutants with pMAD72
3.1. Butanol production by mutants with pCAAD and pSAD42
Plasmid pMTL007 with native thl promoter from C. tyrobutyricum
The wild-type strain (ATCC 25755) does not produce any was used to express adhE2 gene in C. tyrobutyricum for butanol
detectable butanol from glucose because it does not have alde- production. The native thl promoter is a strong and constitutive
hyde/alcohol dehydrogenase genes needed to convert butyryl-CoA promoter and was thus used to control the expression of adhE2
to butanol. Plasmids pCAAD containing aad and its native promo- gene. As can be seen in Fig. 3, the activities of butyraldehyde
ter and pSAD42 containing adhE2 and ptb promoter (see Table 1) dehydrogenase (0.025 U/mg) and butanol dehydrogenase (0.08 U/
from C. acetobutylicum were thus introduced into C. tyrobutyricum mg) of adhE2 increased about 10-fold in the mutant Ct(pMAD72) as
by electroporation, and several mutants were selected and tested compared to those in the wild-type control. Consequently, mutant
for alcohols (ethanol and butanol) production in batch fermenta- Ct(pMAD72) produced 1.1 g/L butanol and 0.14 g/L ethanol in batch
tions with glucose as the substrate. Fig. 2 shows batch fermenta- fermentation while the control (wt with pMTL007 without adhE2)
tions with C. tyrobutyricum mutants Ct(pCAAD) and Ct(pSAD42). produced little or no detectable amounts of butanol and ethanol (see
Butyrate and acetate were two major products in these fermenta- Fig. 4). Compared to the control, the mutant Ct(pMAD72) produced
tions; however, significant amounts of butanol and ethanol were less butyric acid (7.8 vs. 9.7 g/L for the control) and more acetic acid
also produced, confirming that the introduction of aad or adhE2 (2.8 vs. 2.1 g/L for the control). The decreased butyric acid produc-
into C. tyrobutyricum enabled the host cells to produce butanol and tion can be attributed to the conversion of some butyryl-CoA to
ethanol. However, the final butanol titers obtained in these butanol, whereas the slightly increased acetic acid production could
fermentations were low, 67 mg/L in Ct(pCAAD) and 19 mg/L in be due to the need to compensate for the loss of ATP generation
Ct(pSAD42), which could be attributed to the low expression from butyric acid biosynthesis in the mutant.
levels of aad and adhE2 with the heterologous promoters Compared to mutants with pCAAD and pSAD42, mutant
(aad and ptb from C. acetobutylicum) and pIM13 replicon (from Ct(pMAD72) with pMAD72 produced much more butanol
B. subtilis) used in the plasmids pCAAD and pSAD42. The activities (1.1 vs. o0.1 g/L). The higher butanol production can be attrib-
of butyraldehyde dehydrogenase and butanol dehydrogenase in uted to the higher expression level of adhE2 gene with the native,
these mutants were hardly detectable in the mutant cells (data not constitutive thl promoter and higher copy number and better
segregational stability of pMTL007 with replicon pCB102 from
Clostridium butyricum. The segregational stability of pCAAD and
50 12 0.6
pSOS94, both used replicon pIM13 from B. subtilis, was low (only
Ct(pCAAD) Glucose 78% per generation vs. 99% per generation for pMTL007) in
OD600, Acetate & Butyrate (g/L)

Butanol 10 0.5 C. tyrobutyricum. We did not determine the plasmid copy number
Ethanol & Butanol (g/L)

40 Acetic acid
Butyric acid of replicon pIM13 and pCB102 in C. tyrobutyricum. However, we
OD600 found that more E. coli colonies were obtained with replicon
8 0.4
Glucose (g/L)

30 pCB102 than that with pIM13 from plasmid rescue experiments,

6 0.3 suggesting that the plasmid copy number of replicon pCB102 was
20 higher than that of pIM13 in C. tyrobutyricum.
4 0.2

10 3.3. Butanol production in ack and ptb knockout mutants with

2 0.1 pMAD72

0 0 0.0
0 20 40 60 80 100 120 140 160 180 200 Butanol production by ack and ptb knockout mutants of
C. tyrobutyricum expressing adhE2 was investigated. The fermentation
Time (h)
kinetics with these mutants are also shown in Fig. 4. With ptb
knockout, mutant PtbKO(pMAD72) produced 1.7 g/L butanol, a 55%
50 12 0.6
OD600, Acetic & Butyric acid (g/L)

Ct(pSAD42) Butyric acid 0.1

OD600 10 0.5 Ct(pMTL007)
40 Acetic acid
Ethanol & Butanol (g/L)

Enzyme activity (U/mg)

Ethanol 0.08 Ct(pMAD72)

8 0.4
Glucose (g/L)

6 0.3
4 0.2 0.04

10 0.02
2 0.1

0 0 0.0 0
0 20 40 60 80 100 120 140 160 180 200 Butyraldehyde Butanol
Time (h) dehydrogenase dehydrogenase

Fig. 2. Fermentation kinetics of C. tyrobutyricum mutants with plasmid pCAAD Fig. 3. Comparison of butyraldehyde dehydrogenase and butanol dehydrogenase
and pSAD42 in serum bottles with pH controlled at 5.0–6.5 with NaOH. activities of C. tyrobutyricum mutant Ct(pMAD72) expressing adhE2 and
(A) Ct(pCAAD) and (B) Ct(pSAD42). Data are mean 7 s.d. (n¼ 3). Ct(pMTL007). Data are mean 7 s.d. (n¼3).
M. Yu et al. / Metabolic Engineering 13 (2011) 373–382 377

50 12 50 12
Ct(pMTL007) Glucose OD600 Glucose OD600
Butanol Butyric acid
Ct(pMAD72) Butanol Butyric acid
Ethanol Acetic acid Ethanol Acetic acid
10 10
40 40

Products (g/L); OD600

Products (g/L); OD600

8 8
Glucose (g/L)

Glucose (g/L)
30 30

6 6

20 20
4 4

10 10
2 2

0 0 0 0
0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200
Time (h) Time (h)

50 60 12
Glucose OD PtbKO(pMAD72) Glucose OD600
AckKO(pMAD72) Butanol Butyric acid 12
Butanol Butyric acid
Ethanol Acetic acid Ethanol Acetic acid
50 10
Products (g/L); OD600

Products (g/L); OD600

40 8
Glucose (g/L)

Glucose (g/L)

30 8

30 6
20 4

2 10 2

0 0 0 0
0 40 80 120 160 200 240 280 320 360 400 0 40 80 120 160 200 240 280
Time (h) Time (h)

Fig. 4. Fermentation kinetics of C. tyrobutyricum mutants with plasmid pMAD72 in serum bottles with pH controlled at 5.0–6.5 with NaOH. (A) Ct(pMTL007);
(B) Ct(pMAD72); (C) AckKO(pMAD72) and (D) PtbKO(pMAD72). Data are mean 7 s.d. (n¼ 3).

increase from 1.1 g/L by Ct(pMAD72). The increase in butanol fermentation results are also shown in Fig. 4. Compared to
production was expected as less butyryl-CoA was converted to Ct(pMAD72), AckKO(pMAD72) produced much more butanol
butyric acid because of the ptb knockout. However, butyric acid (10.0 vs. 1.1 g/L) and ethanol (0.7 vs. 0.14 g/L) and less
production was still high (9.2 vs. 9.7 g/L by Ct(pMTL007) and 7.8 g/L butyric acid (5.8 vs. 7.8 g/L) and acetic acid (0.22 vs. 2.8 g/L).
by Ct(pMAD72). This is because ptb knockout cannot completely shut The reduced acetic acid production in AckKO(pMAD72) can be
down butyric acid biosynthesis due to the presence of CoA transferase attributed to the blocking of acetate biosynthesis pathway by ack
(see Fig. 1). Compared to Ct(pMAD72), more acetic acid (5.3 g/L) and knockout, which also resulted in increased butanol production
ethanol (0.2 g/L) were produced in PtbKO(pMAD72), indicating that because of increased carbon flux toward butyryl-CoA. As
more carbon substrates were directed towards C2 products as the expected, AckKO(pMAD72) produced more C4 products (15.8 g/L
result of ptb knockout. The results are consistent with our previous butanol and butyric acid) and less C2 products (0.29 g/L acetic
finding that ptb knockout mutant produced more acetate but a acid and ethanol) than did Ct(pMAD72) (8.9 g/L C4 products and
similar amount of butyrate from glucose with a lower butyrate/ 2.94 g/L C2 products) and PtbKO(pMAD72) (10.9 g/L C4 products
acetate ratio as compared to the wild-type (Zhang, 2009). It should be and 5.5 g/L C2 products) because of more carbon flux toward
noted that PtbKO(pMAD72) had a higher growth rate with more butyryl-CoA than toward acetyl-CoA in AckKO(pMAD72). Also,
biomass formation because more energy (ATP) can be produced in AckKO(pMAD72) had a significantly lower specific growth rate
the acetic acid biosynthesis pathway. (see Table 2) because reduced acetate and butyrate production
A higher butyrate/acetate ratio with higher butyrate titer and resulted in less ATP generation.
yield were obtained with C. tyrobutyricum mutant with ack Table 2 summarizes the fermentation results from all mutants.
knockout (Liu et al., 2006b). This mutant was thus studied as It is clear that ack and ptb genes play important roles in controlling
the host to express adhE2 for butanol production, and the the carbon flux distribution in C. tyrobutyricum. For AckKO
378 M. Yu et al. / Metabolic Engineering 13 (2011) 373–382

Table 2
Comparison of cell growth and final product concentrations from various mutant strains of C. tyrobutyricum.

Strain Max. OD600 Specific growth rate (h  1) Butanol (g/L) Butyrate (g/L) Ethanol (g/L) Acetate (g/L) C4/C2 ratio (mol/mol)

Ct-WT 3.4 7 0.11 0.2087 0.003 0 9.26 7 0.13 0.117 0.02 2.1 70.12 2.827 0.19
Ct(pCAAD) 3.3 7 0.15 0.189 7 0.004 0.067 70.004 8.45 7 0.30 0.127 0.006 3.2 70.21 1.777 0.12
Ct(pSAD42) 3.2 7 0.10 0.199 7 0.010 0.019 70.002 8.85 7 0.32 0.127 0.006 3.0 70.20 1.97 0.13
Ct(pMTL007) 3.4 7 0.09 0.2067 0.004 0 9.7 7 0.47 0.087 0.02 2.1 70.26 3.07 0.24
Ct(pMAD72) 3.5 7 0.08 0.1807 0.002 1.1 7 0.1 7.8 7 0.29 0.147 0.01 2.8 70.30 2.17 0.28
PtbKO(pMAD72) 4.07 0.10 0.195 7 0.005 1.7 7 0.25 9.2 7 0.36 0.27 0.005 5.3 70.22 1.47 0.06
AckKO(pMAD72) 3.5 7 0.20 0.115 7 0.011 10.0 70.71 5.8 7 0.21 0.77 0.035 0.22 70.035 10.67 0.55

Data are mean 7s.d. (n¼ 3).

Butyric acid Butanol Acetic acid Ethanol


Product distribution




Ct(pMTL007) Ct(pMAD72) AckKO(pMAD72) PtbKO(pMAD72)

Butyric acid Butanol Acetic acid Ethanol


Product yield (g/g)




Ct(pMTL007) Ct(pMAD72) AckKO(pMAD72) PtbKO(pMAD72)

Fig. 5. Product distribution and yields from glucose in various mutants. (A) Product distribution and (B) product yields.

(pMAD72), one mol glucose yielded 0.64 mol butanol and butanol (10.0 g/L) with 66% theoretical yield or 0.27 g/g glucose
0.31 mol butyric acid, together accounting for 95% glucose fer- fermented.
mented. The ratio of its C4 products (butanol and butyric acid) to
C2 products was 10.6, much higher than 3.0 in wild-type and 3.4. Effects of ack and ptb knockouts on butanol production
2.1 in Ct(pMAD72). In contrast, the ratio of C4 to C2 products was
only 1.4 for PtbKO(pMAD72), which produced 0.50 mol butyrate, Compared to Ct(pMAD72), butanol production increased 600%
0.11 mol butanol from one mol glucose fermented. Fig. 5 shows and 27% with ack knockout and ptb knockout, respectively. With
the product distribution and yields from different mutants. ack knockout, more carbon flux was directed toward butyryl-CoA
The mutant AckKO(pMAD72) produced the highest amount of and thus butanol production increased greatly. The knockout of
M. Yu et al. / Metabolic Engineering 13 (2011) 373–382 379

ack also reduced cell growth, which might have also benefited growth decreased  5–20% at 1.0% (v/v) butanol and  30–50% at
butanol production. With ptb knockout, butanol production 1.5% (v/v) butanol. At 2.0% (v/v) butanol, no significant cell
increased because more butyryl-CoA can be converted to butanol. growth was observed for the first 24 h; however, all strains
However, the increase was not as prominent as with ack knockout started to grow after 2 days. These results showed that
because more carbon flux was directed toward acetyl-CoA, C. tyrobutyricum can tolerate up to 1.5% (v/v) butanol without
resulting in increased acetate production. It is noted that neither adaptation and 2.0% (v/v) butanol after adaptation. For compar-
ack nor ptb knockout can completely block acetate or butyrate ison, C. beijerinckii ATCC 55025 was also investigated, and the
biosynthesis in C. tyrobutyricum because of the presence of CoA results showed that its growth rate was reduced  20% and  85%
transferases (unpublished data), which catalyze alternative path- at 1% and 1.5% (v/v) butanol, respectively (Fig. 7E). C. beijerinckii
ways for acetate and butyrate biosynthesis from acetyl-CoA and was completely inhibited by 2% (v/v) butanol as no cell growth
butyryl-CoA, respectively (see Fig. 1). This has also been observed was observed even after 10 days incubation. It is clear that
in other clostridia mutants with inactivated acetate and butyrate C. tyrobutyricum has higher butanol tolerance than solventogenic
pathways (Green et al., 1996; Sillers et al., 2008). Some butyrate- Clostridia, which usually cannot grow at butanol concentrations
producing microbes use the pathway with butyryl-CoA:acetate higher than 1–1.5% (v/v), depending on strains. For example, the
CoA transferase, instead of phosphotransbutyrylase and butyrate specific growth rate of C. acetobutylicum ATCC 824 was reduced
kinase, for butyrate biosynthesis (Duncan et al., 2002). To further by 50% at 1.1% (v/v) butanol and little or no growth was observed
enhance butanol production, it may be necessary to knockout at 1.5% (v/v) butanol (Baer et al., 1987). Although Clostridium
both ack and ptb. It is also of interest to also knockout CoA could acquire higher solvent tolerance via classic adaptation (Baer
transferase and to study its effect on the fermentation. et al., 1987) or metabolic engineering such as overexpressing
stress proteins (Tomas et al., 2003), improvement in butanol
3.5. Effect of substrate reducing state on butanol production production or titer was limited or insignificant. For example,
adaptation resulted in a mutant with 27% increase in butanol
Electron balance plays an important role in cellular metabo- tolerance, but little or no butanol was produced by this mutant
lism and product synthesis. Butanol biosynthesis requires redu- (Baer et al., 1987). Therefore, based on the butanol tolerance,
cing power, usually from NADH, and more butanol could be C. tyrobutyricum appears to be a favorable host for butanol
produced from a more reduced substrate (Bond-Watts et al., production.
2011; Vasconcelos et al., 1994). Fig. 6 shows the fermentation
kinetics of mannitol as the carbon source with mutant AckKO 3.7. Comparison with other engineered mutants for butanol
(pMAD22). As expected, higher butanol titer (  16 vs. 10 g/L) and production
yield (  30.6% vs. 27% w/w) were obtained with lower acetate and
butyrate production ( 1.0 vs. 45 g/L) when the more reduced Recently, there have been intensive efforts to engineer non-
substrate (mannitol vs. glucose) was used in the fermentation. It solventogenic microbes, including E. coli, S. cerevisiae, B. subtilis,
is clear that the more reduced substrate increased NADH avail- P. putida and L. brevis (Atsumi et al., 2008; Berezina et al., 2010;
ability and drove more metabolic flux toward the more reduced Nielsen et al., 2009; Steen et al., 2008), for butanol production
product, butanol, against the more oxidized acid products. because of their potentially higher butanol tolerance and easier to
clone than Clostridia. Some microbes including several strains of
3.6. Butanol tolerance P. putida can tolerate up to 6% (w/v) butanol (Ruhl et al., 2009),
although the butanol toxic threshold is between 1% and 2% (w/v)
Butanol with a four-carbon chain length is more toxic to for most microorganisms (Knoshaug and Zhang, 2009). Most
microbes than ethanol. Butanol tolerance is a critical factor S. cerevisiae strains were tolerant to 1% (v/v) butanol with 60%
limiting cell’s ability to produce butanol. To assess butanol relative specific growth rate, and three strains could grow in 2%
tolerance, the effect of butanol concentration in the culture (v/v) butanol with 10–20% relative growth rate (Knoshaug and
medium on cell growth was studied, and the results are shown Zhang, 2009). For E. coli, its growth rate decreased to 20–40% and
in Fig. 7. In general, 0.5% (v/v) butanol did not have any apparent 40–60% at 1% (v/v) butanol at 37 and 30 1C, respectively, and no
effect on cell growth for all strains studied. At higher butanol growth was observed at 2% (v/v) butanol (Knoshaug and Zhang,
concentrations, significant growth inhibition was observed. Cell 2009). It had been shown that strains with a higher content of
saturated fatty acids in their cytoplasmic membranes, which
contributed to lower membrane fluidity, also had higher mem-
50 20 brane stability and resistance against chaotropic agents such as
Mannitol OD600
butanol (Ezeji et al., 2010; Liu and Qureshi, 2009). However, the
Butanol acetate
butyrate ethanol mechanism and cellular response on butanol stress is quite
40 16
Products (g/L); OD600

complicated and involve many factors including transcription

factors, altered membrane components, efflux pump, stress pro-
Mannitol (g/L)

30 12 teins, energy metabolism and small molecule chaperone, and

chemical detoxification (Nicolaou et al., 2010).
The Clostridia’s solventogenic pathway, that involves multiple
20 8
genes, has been successfully cloned and expressed in several
microorganisms (see Table 3). However, the highest butanol titer
10 4 obtained so far was only 580 mg/L in E. coli (Nielsen et al., 2009),
300 mg/L in L. brevis (Berezina et al., 2010), 120 mg/L in P. putida,
24 mg/L in B. subtilis, (Nielsen et al., 2009), and 2.5 mg/L in
0 0
0 50 100 150 200 250 300 S. cerevisiae (Steen et al., 2008). The slow and reversible turn-over
rate of Clostridium butyryl dehydrogenase complex (bdh) could limit
Time (h)
the butanol production (Bond-Watts et al., 2011; Li et al., 2008).
Fig. 6. Fermentation kinetics of AckKO(pMAD22) with mannitol as the substrate A keto-acid pathway has also been expressed in E. coli for n-butanol
in serum bottles. Data are mean 7s.d. (n¼ 3). production, achieving butanol titer of 800 mg/L (Shen and Liao,
380 M. Yu et al. / Metabolic Engineering 13 (2011) 373–382

5 5
Ct(pMTL007) Ct(pMAD72)
4 0 4 0
0.5 0.5
1 1
3 1.5 3 1.5

2 2

2 2

1 1

0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time, h Time, h

5 5
AckKO(pMAD72) PtbKO(pMAD72)
4 0 4 0
0.5 0.5
1 1
3 1.5 3 1.5


2 2

2 2

1 1

0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time, h Time, h

Relative Growth Rate



0.4 Ct(pMTL007)
0.2 AckKO(pMAD72)
0 0.5 1 1.5 2
Butanol Concentration (v/v %)

Fig. 7. Effects of butanol on the growth of various C. tyrobutyricum mutants. (A) Ct(pMTL007); (B) Ct(pMAD72); (C) AckKO(pMAD72); (D) PtbKO(pMAD72) and (E) Relative
specific growth rates of various mutants and C. beijerinkii ATCC 55025. Data are mean 7 s.d. (n¼3).

2008). Recently, a higher n-butanol titer of 4.65 g/L was obtained could restore butanol and ethanol production in M5, with butanol
using a chimeric pathway assembled from three different organisms titers reaching 5.5 and 11.1 g/L, respectively (Fontaine et al.,
(Ralstonia eutrophus pha, C. acetobutylicum hbd, crt, and Treponema 2002; Sillers et al., 2008). We determined that the low butanol
denticola ter replacing bdh) and overexpressing native pyruvate titer in our mutants Ct(pCAAD) and Ct(pSAD42) was caused by
dehydrogenase complex (aceEF.lpd) in E. coli (Bond-Watts et al., the low expression efficiency and poor plasmid stability due to
2011). Nevertheless, none of these heterologous hosts can produce the heterologous promoters and replicon used in the expression
n-butanol at a titer comparable to that from solventogenic Clostri- plasmids. Using plasmid pMTL007 and the native thl promoter,
dia, which usually produce 12–16 g/L butanol in ABE fermentation we achieved a butanol titer of 1.1 g/L, which is much higher than
(Papoutsakis, 2008). those from recombinant E. coli and all other heterologous hosts
In this study, we were able to make the non-solventogenic ever reported in the literature. By using the ack knockout mutant
C. tyrobutyricum to produce butanol by cloning just one gene of C. tyrobutyricum as the host, the butanol titer was further
adhE2. Our initial attempts with plasmids pCAAD and pSAD42 increased to 10 g/L, which is comparable to the level from
were not satisfactory, although introducing pCAAD into the non- solventogenic clostridia. In addition, higher butanol titer (16 g/L)
solventogenic mutant strain M5 of C. acetobutylicum, which had and yield (30.6%) were obtained from a more reduced substrate,
lost the megaplasmid containing all solventogenic genes, could mannitol.
restore its ability to produce butanol and ethanol (Nair and Further improvements of our C. tyrobutyricum mutants are
Papoutsakis, 1994). Also, expressing adhE2 gene under the control possible. Although our mutants expressing adhE2 showed good
of thl promoter and aad gene under the control of ptb promoter butyraldehyde dehydrogenase and butanol dehydrogenase activities
M. Yu et al. / Metabolic Engineering 13 (2011) 373–382 381

Table 3
Comparison of n-butanol production in various metabolically engineered hosts.

Host Gene expression Gene knockout Butanol titer (g/L) Reference

E. coli thrAfbrBC, kivd, ADH2, ilvA, leuABCD DmetA, Dtdh, DilvB, DilvI, DadhE 0.8 Shen and Liao (2008)
E. coli crt, bcd, etfAB, hbd, atoB, adhE2 DadhE, DldhA, DfrdBC, Dfnr, Dpta 0.55 Atsumi et al. (2008)
E. coli crt, bcd, etfAB, hbd, atoB, adhE2, gapA 0.58 Nielsen et al. (2009)
E. coli pha, hbd, crt, ter, adhE2, aceEF.lpd 4.65 Bond-Watts et al. (2011)
B. subtilis crt, bcd, etfAB, hbd, thl, adhE2 0.024 Nielsen et al. (2009)
P. putida crt, bcd, etfAB, hbd, thl, adhE1 0.12 Nielsen et al. (2009)
S. cerevisiae ERG10, hbd, crt, ccr, adhE2 0.0025 Steen et al. (2008)
L. brevis crt, bcd, etfAB, hbd 0.3 Berezina et al. (2010)
C. acetobutylicum M5 adhE2 5.5 Fontaine et al. (2002)
C. acetobutylicum M5 aad 11.1 Sillers et al. (2008)
C. tyrobutyricum adhE2 Dack 10.0–16.0a This study

Genes from E. coli: aceEF.lpd (pyruvate dehydrogenase complex), adhE (fused acetaldehyde-CoA dehydrogenase/iron-dependent alcohol dehydrogenase/pyruvate-formate
lyase deactivase), atoB (acetyl-CoA acetyltransferase), fnr (DNA-binding transcriptional dual regulator, global regulator of anaerobic growth), frdBC (fumarate reductase
Fe-S subunit and membrane anchor subunit), gapA (glyceraldehyde-3-phosphate dehydrogenase A), ilvA (threonine deaminase), ilvB (acetolactate synthase I large subunit),
ilvI (acetolactate synthase III, large subunit), ldhA (D-lactate dehydrogenase), leuA (2-isopropylmalate synthase), leuB (3-isopropylmalate dehydrogenase), leuCD (NAD(þ )-
dependent 3-isopropylmalate dehydratase), metA (homoserine O-transsuccinylase), pta (phosphate acetyltransferase), tdh (threonine 3-dehydrogenase), thrAfbr (feedback
resistant fused aspartokinase I and homoserine dehydrogenase I), thrBC (homoserine kinase and threonine synthase).
Genes from C. acetobutylicum: aad, adhE1 (alcohol/aldehyde dehydrogenase 1), ack, crt, bcd, etfAB, hbd, thl, adhE2 (see Fig. 1).
Other genes: ADH2 (alcohol dehydrogenase 2 from S. cerevisiae); ccr (Butyryl-CoA dehydrogenase from Streptomyces collinus); ERG10 (acetoacetyl-CoA thiolase from
S. cerevisiae); Kivd (2-ketoacid decarboxylase from Lactococcus lactis); pha (acetoacetyl-CoA thiolase from Ralstonia eutrophus); ter (NADH dependent crotonyl-CoA specific
trans-enoyl-CoA reductase from Treponema denticola).
Using mannitol as substrate.

(0.025 and 0.08 U/mg, respectively), they were lower than those in theoretical yield of 0.41 g/g glucose could be achieved in
recombinant E. coli (0.051 and 0.13 U/mg) (Inui et al., 2008) and C. C. tyrobutyricum by blocking its butyrate synthesis pathway
acetobutylicum DSM 1732 (0.038 and 0.14 U/mg) (Durre et al., 1987). (knockout ptb or buk), increasing adhE2 expression level using a
Furthermore, C. acetobutylicum M5 with adhE2 expression showed a stronger promoter, and increasing NADH availability by rebalan-
much higher butyraldehyde dehydrogenase activity of 0.3 U/mg cing the redox potential through engineering hydrogenases and
(Fontaine et al., 2002). High copy number expression plasmids with expressing heterologous pathway for NADH formation (Berrios-
stronger constitutive or regulatory promoters could be used to Rivera et al., 2002; Nielsen et al., 2009; Zhou et al., 2010).
improve adhE2 expression in C. tyrobutyricum.
In addition, it has been shown that changing NADH/NAD þ
ratio redistributed metabolic flux and product formation in E. coli Acknowledgments
(Berrios-Rivera et al., 2002) and C. acetobutylicum (Vasconcelos
et al., 1994). Down-regulation of hydrogen-uptake hydrogenase, This work was supported by the National Science Foundation
which converts hydrogen to NAD(P)H, resulted in a low butanol STTR program (IIP-0810568, IIP-1026648) and Ohio Department
and high acetone production in C. saccharoperbutylacetonicum of Development—Third Frontier Advanced Energy Program (Tech
(Nakayama et al., 2008). Butanol concentration was improved 08-036). We would like to thank Prof. N.P. Minton, University of
1.6-fold via increasing NADH availability by overexpressing pyr- Nottingham, UK for providing the donor E. coli CA434 and plasmid
uvate decarboxylase complex in E. coli (Bond-Watts et al., 2011). pMTL007, Prof. G.N. Bennett, Rice University, for plasmid pCAAD
Our results from mannitol also suggested that increasing NADH and Prof. E.T. Papoutsakis, University of Delaware, for plasmid
can enhance butanol titer and yield. Therefore, butanol biosynth- pSOS94.
esis in C. tyrobutyricum could be further enhanced by overexpres-
sing formate dehydrogenase or pyruvate dehydrogenase.
In summary, C. tyrobutyricum is a good heterologous host for Appendix A. Supplementary material
n-butanol production. In addition to its higher butanol tolerance,
C. tyrobutyricum as a native acidogen is not tightly regulated Supplementary data associated with this article can be found
when used for butanol production, and thus would be easier to in the online version at doi:10.1016/j.ymben.2011.04.002.
manipulate genetically and to control during fermentation. Con-
ventional solventogenic Clostridia with complex metabolic and
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