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Research review paper

Engineering of microorganisms for the production of biofuels and perspectives based

on systems metabolic engineering approaches
Yu-Sin Jang
, Jong Myoung Park
, Sol Choi
, Yong Jun Choi
, Do Young Seung
Jung Hee Cho
, Sang Yup Lee
a, b,

Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), BioProcess Engineering Research Center,
Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, Daejeon, Republic of Korea
Department of Bio and Brain Engineering and Bioinformatics Research Center, KAIST, Daejeon, Republic of Korea
GS Caltex Corporation Value Creation Center, Daejeon, Republic of Korea
a b s t r a c t a r t i c l e i n f o
Available online xxxx
Systems metabolic engineering
Systems biology
Synthetic biology
Microbial cell factory
The increasing oil price and environmental concerns caused by the use of fossil fuel have renewed our inter-
est in utilizing biomass as a sustainable resource for the production of biofuel. It is however essential to de-
velop high performance microbes that are capable of producing biofuels with very high efciency in order to
compete with the fossil fuel. Recently, the strategies for developing microbial strains by systems metabolic
engineering, which can be considered as metabolic engineering integrated with systems biology and synthet-
ic biology, have been developed. Systems metabolic engineering allows successful development of microbes
that are capable of producing several different biofuels including bioethanol, biobutanol, alkane, and biodie-
sel, and even hydrogen. In this review, the approaches employed to develop efcient biofuel producers by
metabolic engineering and systems metabolic engineering approaches are reviewed with relevant example
cases. It is expected that systems metabolic engineering will be employed as an essential strategy for the de-
velopment of microbial strains for industrial applications.
2011 Elsevier Inc. All rights reserved.
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. Metabolic engineering of microorganisms for the production of biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1. Considerations on strain development for the production of biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2. Ethanol producers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.3. Butanol and higher alcohol producers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.4. Gasoline and diesel producers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.5. Algal fuel and hydrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.6. Isoprenoid-based fuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Future perspectives on systems metabolic engineering for biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3.1. Tools and approaches employed systems metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3.2. Production of biofuels using systems metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Biotechnology Advances xxx (2011) xxxxxx
Corresponding author at: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Tel.: +82 42 350
3930; fax: +82 42 350 3910.
E-mail address: (S.Y. Lee).
JBA-06478; No of Pages 12
0734-9750/$ see front matter 2011 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
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Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
1. Introduction
Due to the limited nature of fossil resources, oil price is vulnerable
to many factors and is expected to increase more rapidly fromnowon
(Kerr, 2007). Recent sharp increase in oil price caused by the turmoil
in north African countries proves that this is the case. Also, climate
change issue is becoming more and more serious, and thus there
has been a global movement toward reduced use of fossil resources.
Thus, bio-based production of fuels and chemicals from renewable
biomass is becoming increasingly attractive and will be essential for
our future. Bioethanol and biodiesel are already commercialized as al-
ternative fuels in the market worldwide. Bio-based production of bu-
tanol and alkane is also being pursued around the world. It is clear
that biofuels will be increasingly used to replace some of fossil fuel
for our sustainable future (Farrell et al., 2006; Lynd et al., 2008).
Inorder to efciently produce fuels fromrenewable resources, micro-
organisms having superior metabolic capabilities need to be developed.
Traditionally microbial strains for industrial applications have been de-
veloped by the random mutagenesis and screening processes (Labarre
et al., 2001). However, this approach results in unknown genotypic/
phenotypic changes and can cause unwanted alterations in the genome
that might become problematic when production condition needs to
be modied; further metabolic engineering of the random mutant cells
is often difcult (Park et al., 2008). Consequently, rational metabolic en-
gineering has become a standard strategy for strain development over
the last couple of decades (Lee and Papoutsakis, 1999; Stephanopoulos
et al., 1998). However, these approaches also attained to some limitations
in order to improve cellular performances dramatically because the
scope of engineering cells is often local rather than system-wide.
Recently, systems metabolic engineering has been developed toward
engineering the organism at the systems-level beyond traditional
metabolic engineering (Lee et al., 2005a; Palsson and Zengler, 2010).
Systems metabolic engineering has been employed for developing
strains for the production of bioproducts, including biofuels (Jarboe
et al., 2007; Lee et al., 2009; Mukhopadhyay et al., 2008; Otero et
al., 2007; Ranganathan and Maranas, 2010; Schirmer et al., 2010;
Steen et al., 2010; Tang and Zhao, 2009). Several techniques including
high-throughput screening, in silico modeling and simulation, omics
(genome, transcriptome, proteome, metabolome, and uxome), gene
synthesis, and synthetic regulatory circuits, and enzyme and pathway
engineering have been employed during systems metabolic engineer-
ing (Kim et al., 2008a; Lee et al., 2005b; Lee and Papoutsakis, 1999;
Palsson and Zengler, 2010; Park et al., 2008, 2009). Here, we review
several recent case studies of metabolic engineering for the produc-
tion of biofuels, and suggest future perspectives on successful strain
development using a more rened version of metabolic engineering,
namely systems metabolic engineering.
2. Metabolic engineering of microorganisms for the production
of biofuels
For industrial applications of microorganisms for the production
of desired biofuels, the metabolic performance needs to be improved
by metabolic engineering. There have recently been reports on the
Fig. 1. Summary of key approaches taken to the development of strains for the production of biofuels including ethanol, butanol, gasoline, diesel, and algal fuels, which are indicated
as gray ovals. Small circles located within each oval together with references indicate approaches taken for strain improvement: random mutagenesis, rational engineering, omics,
in silico analysis, and synthetic biology; these small circles are arranged in a way that they are grouped to large circles in the background.
2 Y.-S. Jang et al. / Biotechnology Advances xxx (2011) xxxxxx
Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
use of metabolically engineered microorganisms for the production of
several different biofuels. General strategies for metabolic engineer-
ing of microorganisms and specic examples on the use of these strat-
egies for the production of biofuels are described below, and
summarized in Fig. 1. Some examples of systems metabolic engineer-
ing approaches are also discussed in next section with future perspec-
tives, and summarized in Table 1.
2.1. Considerations on strain development for the production of biofuels
Constructing microorganisms toward desired fuel production should
take into account several considerations, including enhancement of prod-
uct concentration, yield and productivity, simplication of downstream
processes, and utilization of inexpensive substrates, which are dis-
cussed in next sections. Besides, the control of redox balance and
the fuel tolerance also deserve attention for enhanced fuel production.
Control of redox balance is also one of the most important obstacles in
strain development for the production of fuels at high yields. In general,
high redox potential is needed to efciently produce ethanol and buta-
nol, which are all relatively reduced products. To increase the yield
andproductivity, cofactor manipulationhas beenappliedas anessential
and powerful tool for metabolic engineering. For examples, over-
expression of the NAD
-dependent Fdh doubled the maximum yield
of NADHfrom2 to 4 mol NADH/mol glucose consumed, resulting in in-
creased cell density and ethanol production (Berrios-Rivera et al.,
2002). As a result, ethanol production by Escherichia coli could be
Table 1
Representative systems metabolic engineering strategies of the strain development for the production of biofuels.
Fuels Strain Strategies for systematic approach References
Ethanol S. cerevisiae
E. coli
Z. mobilis
Construction of genome-scale metabolic models
for ethanologenic organisms
Feist and Palsson (2008); Lee et al.
(2010); Mo et al. (2009)
Ethanol S. cerevisiae Genome-scale metabolic model of S. cerevisiae aided the
improvement of ethanol production to increase ethanol
yield and decrease glycerol production under anaerobic
conditions using glucose as a carbon source
Bro et al. (2006); Hjersted
et al. (2007)
Ethanol E. coli OptReg method has suggested the optimal metabolic
engineering strategies of activation, repression, and
elimination of reactions for the overproduction of ethanol
Pharkya and Maranas (2006)
Ethanol and higher alcohols E. coli OptORF method has been developed to identify optimal gene
knockout and amplication targets by incorporating metabolic
networks with transcriptional regulatory networks; employed
for the production of ethanol and higher alcohols
Kim and Reed (2010)
Ethanol E. coli Transcriptome analysis to identify expression changes on
ethanologenic E. coli; Identifying both glycine and betaine can
serve as protective osmolytes
Gonzalez et al. (2003)
Butanol C. acetobutylicum
C. beijerinckii
Genome sequencing of clostridia, which has given chance
more systematic approaches for metabolic engineering
of butanol producers
Nolling et al. (2001)
Butanol C. acetobutylicum In silico genome-scale metabolic models have been
reconstructed, which allow genome-scale ux analysis
and other simulations for designing the metabolic
engineering strategies
Lee et al. (2008a); Senger and
Papoutsakis (2008a,2008b)
Butanol E. coli OptForce has considered the change of computational
and possible ux ranges for all metabolic reactions in
bioproduct-overproducing networks compared with
the wild type metabolic network; used in the increased
production of butanol
Ranganathan et al. (2010); Ranganathan
and Maranas (2010)
Butanol Computational algorithms have been suggested to predict
metabolic pathways based on chemical structure; used to
predict butanol production pathway
Cho et al. (2010); Li et al. (2004)
Butanol C. acetobutylicum Genome-wide transcriptome and proteome analysis; used
in studies on stress response and sporulation; stress response
genes (groES, dnaKJ, hsp18, and hsp90) have been identied
Alsaker et al. (2004, 2010); Jones et al.
(2008); Mao et al. (2010); Schwarz et al.
(2007); Sullivan and Bennett (2006);
Tomas et al. (2003a,2003b, 2004)
Butanol C. acetobutylicum
E. coli
Genomic library screening of C. acetobutylicum and E. coli to
improve butanol tolerance in genome-wide manner; CAC0003
and CAC1869 (in C. acetobutylicum) and the entC and feoA
(in E. coli) have been identied to increase butanol tolerance
Borden and Papoutsakis (2007); Reyes
et al. (2011)
Butanol C. acetobutylicum Genome-wide non-coding RNAs study; RDNA7 increased
butyrate tolerance of C. acetobutylicum
Borden et al. (2010)
Isobutanol E. coli Genome-wide systems analyses; whole genome sequencing
followed by gene repair and knockout study; genes
(acrA, gatY, tnaA, yhbJ, and marCRAB) related to
isobutanol tolerance have been identied
Atsumi et al. (2011)
Isobutanol E. coli Evolution combined with genomic study; genes
(marC, hfq, mdh, acrAB, gatYZABCD and rph) lead
increased tolerance to isobutanol
Minty et al. (2011)
Alternative biofuels including
geraniol, geranyl acetate,
limonene, and farnesyl
E. coli Identication of novel biofuel efux pumps from sequenced
bacterial genomes based on bioinformatics; identied library
of 43 pumps has been heterologously expressed in E. coli
and tested by using a comparative growth assay; some
identied pumps have restored growth in the presence
of biofuels including geraniol, geranyl acetate, limonene,
and farnesyl hexanoate; none of the pumps have been
identied for improving tolerance to butanol and isopentanol
Dunlop et al. (2011)
3 Y.-S. Jang et al. / Biotechnology Advances xxx (2011) xxxxxx
Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
dramatically increased by 22-fold relative to the control strain under
anaerobic condition (Berrios-Rivera et al., 2002). In another example,
metabolic engineering was performed to increase NADH level in E. coli
by disrupting the ldh, adh, and frd genes, while the Candida boidinii
fdh gene was overexpressed. This metabolic engineering strategy
resulted in the construction of E. coli strain having capability to produce
15 g/L of butanol (Shen et al., 2011).
Achieving hightiters of target fuels is closely related to the endprod-
uct stress to the host cell (Taylor et al., 2008). The cellular stress to these
biofuels can be reduced by amplication of tolerance related-proteins
or addition of protectant in the culture media, which often increases
the nal titer of the target product. One approach to identify target
genes to be manipulated for increasing tolerance against ethanol is
transcriptome analysis, which is discussed in the last section. Another
approach to increase the product tolerance is directed or adaptive evo-
lution. The E. coli EMFR9 strain could be evolved to be able to grow in
the presence of furfural at high concentration (Miller et al., 2009). Sim-
ilarly, Saccharomyces cerevisiae M25 strain evolved with transcription
factors could become more tolerant to ethanol (Zhao et al., 2010).
More recently, butanol tolerance of E. coli could be increased by using
articial transcription factor (ATF) libraries which consist of zinc nger
DNA-binding proteins and E. coli cyclic AMP receptor protein (Lee et al.,
2011b). The selected butanol-tolerant E. coli strain could grow in the
presence of up to 1.5% (v/v) of butanol (Lee et al., 2011b).
2.2. Ethanol producers
Ethanol is currently the only renewable biofuel that is produced at
sufciently large scale, and used as an additive or alternative for gas-
oline. The amount of worldwide ethanol production for transport fuel
is continuously increasing (Bringezu et al., 2009). It is forecasted that
the global use of biofuel including bioethanol is expected to nearly
double in 2017 (Bringezu et al., 2009; RFA, 2010). The United States
and Brazil produced and used most ethanol for transportation, as
much as 89% of the worldwide ethanol production in 2009, and man-
dated the blending ethanol with gasoline (RFA, 2010; Worldwatch,
2006). In order to achieve higher yield and productivity due to the
importance of ethanol, many different metabolic engineering studies
have been performed.
S. cerevisiae and Zymomonas mobilis are native ethanol producers
that can efciently convert glucose to ethanol (Fig. 2A), but cannot
use pentose sugars as carbon sources (Almeida et al., 2011; Matsushika
et al., 2009; Seo et al., 2005). Because of the concerns on the possible in-
crease of food prices due to the use of edible biomass for ethanol
Fig. 2. Major pathways for the production of (A) ethanol, (B) butanol, (C) biodiesel, (D) algal fuel, and (E) fuels fromisoprenoid. Abbreviations: PDC, pyruvate decarboxylase; ADH, alcohol
dehydrogenase; PFL, pyruvate formate lyase; ACTDH, acetaldehyde dehydrogenase; PFOR, pyruvate:ferredoxin oxidoreductase; THL, thiolase; HBD, 3-hydroxybutyryl-CoA dehydroge-
nase; CRT, crotonase; BCD, butyryl-CoA dehydrogenase; AAD, butyraldehyde dehydrogenase; BDH, butanol dehydrogenase; TES, thioesterase; FADD, fatty acyl-CoA synthase; FAR,
fatty acyl-CoA reductase; WS/DGAT, acyltransferase; HMG-CoA, hydroxymethylglutaryl-CoA; MEV, mevalonate; IPP, isopentenyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate;
MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-MEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; HMB-PP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; DMAP,
dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; AtoB, acetoacetyl-CoA thiolase; HMGS, hydroxymethylglutaryl-
CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, diphosphomevalonate decarboxylase; DXS, 1-deoxy-D-
xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; CMS, C-methyl-erythritol cyclodiphosphate synthase; CMK, C-methyl-erythritol kinase;
MCS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, hydroxy-methylbutenyl diphosphate synthase; HDR, hydroxy-methylbutenyl diphosphate reductase; GPPS, geranyl
diphosphate synthase; FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; PPI, diphosphate.
4 Y.-S. Jang et al. / Biotechnology Advances xxx (2011) xxxxxx
Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
production, lignocellulosics have been generally considered as a good
carbon source. In contrast to S. cerevisiae and Z. mobilis, E. coli can uti-
lize most carbohydrate components present in lignocellulosics but
produce only a small amount of ethanol during fermentation (Neid-
hardt et al., 1996). Accordingly, E. coli strains have been metabolically
engineered for enhanced ethanol production through the introduction
of foreign genes, elimination of competitive pathways, and disruption
of byproducts formation (Jarboe et al., 2007). The resulting strain, E.
coli KO11, was constructed based on E. coli W stain by introducing for-
eign genes encoding pyruvate decarboxylase and alcohol dehydroge-
nase (PET operon) from Z. mobilis and disrupting fumarate reductase
(Jarboe et al., 2007). The E. coli KO11 strain was able to produce eth-
anol at about 95% of the theoretical yield in a complex medium, which
is as good as S. cerevisiae. In addition, to increase ethanol tolerance in
a complex medium, the LY01 strain was constructed by adaptive evo-
lution and selection. However, the dependence of both E. coli KO11
and LY01 on complex nutritional supplement increased the cost of
ethanol production. Thus, a new ethanologenic E. coli strain starting
from SZ110 strain, a derivative of KO11, was constructed to improve
the yield of ethanol production in minimal medium (Zhou et al.,
2005). The E. coli SZ110 strain was redesigned to produce lactic acid
by removing PET operon, alcohol dehydrogenase, and acetate kinase
from E. coli KO11 strain. For ethanol production in minimal medium,
the SZ110 strain was reprogrammed again by eliminating lactate de-
hydrogenase and inserting pyruvate formate lyase and PET operon
from Z. mobilis (Jarboe et al., 2007). The resulting strain, LY168,
could produce 0.5 g ethanol per gram of xylose, which is close to
the theoretical maximum yield of 0.51, in a minimal medium contain-
ing betaine. Furthermore, a homo-ethanol producer, SE2378 (ldh
p), which is constructed by mutagenesis, was able to produce eth-
anol with the productivity of 2.24 g/h/g-cells at approximately 80% of
the theoretical ethanol yield (Kim et al., 2007). About 88% of fermen-
tation metabolites produced by SE2378 was ethanol. Another homo-
ethanol producer, SZ420 (frdBC ldh ackA folA-p pdhR::
pBp6-aceEF-lpd), which is constructed by eliminating the competing
fermentation pathways in E. coli B and highly expressing pyruvate de-
hydrogenase complex (aceEF-lpd, a typical aerobically-expressed op-
eron) under anaerobic condition, could achieve a 90% ethanol yield
from xylose (Zhou et al., 2008). More recently, an adaptive evolution
of nontransgenic E. coli KC01 (ldhA pB ackA frdBC pdhR::pBp6-
aceEF-lpd) has been performed to achieve homo-ethanol production,
which results in improvement of ethanol production with 94% yield
from xylose (Wang et al., 2010b).
2.3. Butanol and higher alcohol producers
Butanol, a four carbon primary alcohol (C
O), has recently
been attracting much interest due to its great potential to be used
as an alternative fuel in addition to its existing applications as a sol-
vent (Durre, 2007, 2008; Lee et al., 2008b; Papoutsakis, 2008). It has
been estimated that 1012 billion pounds of butanol is produced an-
nually, which accounts for 78.4 billion dollar market at current price
(Lee et al., 2008b). It is notable that butanol and higher alcohols can
be used as a direct replacement of gasoline or as a fuel additive. Buta-
nol can be used directly in any gasoline engine without modication
and/or substitution, because it has sufciently similar characteristics
to gasoline as a liquid fuel. For example, in 2005, a gasoline car with
unmodied engine was fueled with 100% butanol and successfully
ran almost 10,000 miles across the USA. This result clearly demon-
strates that biobutanol is one of the most powerful alternative fuel.
Clostridium (Durre, 2008; Jiang et al., 2009; Lee et al., 2009; Qureshi
et al., 2008; Sillers et al., 2008) and some engineered E. coli (Atsumi
et al., 2008a,2008b) strains are two popular butanol (or isobutanol)
producers being studied intensively.
One of the characteristics of the butanol-producing clostridia is bi-
phasic fermentation (Lee et al., 2008b). During the rst phase, acetate
and butyrate are produced as major products and pH goes down
below 5.0, which is known as acidogenic phase. Then solventogenic
phase follows, during which acetate and butyrate are reassimilated
to form solvents, butanol, acetone and ethanol. The major objectives
of metabolic engineering of clostridia include efcient butanol pro-
duction with respect to the titer, yield, and selectivity of butanol. Be-
fore genome sequencing of clostridia, several interesting examples of
random mutagenesis and rational metabolic engineering of clostridia
have been reported (Bennett and Rudolph, 1995; Formanek et al.,
1997; Green et al., 1996; Green and Bennett, 1998). C. beijerinckii
BA101 strain was isolated from the mutant pool of C. beijerinckii
NCIMB 8052 treated with a mutagen N-methyl-N'-nitro-N-nitroso-
guanidine (NTG), and selected on nonmetabolizable glucose analog,
2-deoxyglucose (Formanek et al., 1997). C. beijerinckii BA101 is able
to produce higher level of butanol (18.6 g/L) than its parent strain
(9.2 g/L).
There have been several metabolic engineering approaches taken
to improve butanol production in C. acetobutylicum. The PJC4BK strain
was constructed by the disruption of the buk gene encoding butyrate
kinase involved in butyrate formation pathway in C. acetobutylicum
(Green et al., 1996). This strain was able to produce butanol up to
16.7 g/L, which is much higher than that (11.7 g/L) obtained with
the wild-type strain (Harris et al., 2000). Another engineering ap-
proach taken was increasing the butanol titer through enhancing
the end-products tolerance. Overexpression of the molecular chaper-
one GroESL, which allowed the solventogenic enzymes to be in more
active states, resulted in an increase of the nal solvent titer (Tomas
et al., 2003b).
High butanol selectivity is important as it can reduce the recovery
cost (Jiang et al., 2009; Lee et al., 2009). C. acetobutylicum M5 strain is
a derivative of C. acetobutylicum ATCC 824, and it does not produce
butanol due to the lack of the megaplasmid pSOL1 (Clark et al.,
1989). C. acetobutylicum M5 was metabolically engineered to increase
the butanol selectivity to total solvents (Lee et al., 2009; Sillers et al.,
2008). The adhE1 gene was overexpressed under the control of the
ptb promoter in the M5 strain, which restored butanol production
to the level that can be achieved with ATCC 824 while without pro-
ducing acetone (Sillers et al., 2008). The introduction of the adhE1-
ctfAB genes into the M5 strain also increased the butanol selectivity
(Lee et al., 2009). The metabolically engineered C. acetobutylicum
M5(pIMP1E1AB) was able to produce butanol with high butanol se-
lectivity to total solvents (0.84), which is much higher than that
(0.57) typically obtained with the ATCC 824 strain. In another study,
the butanol selectivity could be increased to 0.82 by disrupting the
adc gene encoding acetoacetate decarboxylase in C. acetobutylicum
and regulating electron ow by the addition of methyl viologen into
culture medium (Jiang et al., 2009).
It should be noted that genetic manipulations such as gene am-
plication and gene knockout, particularly in native butanol produc-
er Clostridium sp., is extremely difcult. Due to this reason, E. coli
and yeast have been attracted as alternative hosts for butanol pro-
duction. Introduction of clostridial genes including thl, hbd, crt,
bcd, etfAB and adhE2, responsible for butanol formation in C. aceto-
butylicum (Fig. 2B), resulted in the production of 139 mg/L of buta-
nol in E. coli under anaerobic condition (Atsumi et al., 2008a).
Butanol production by the metabolically engineered E. coli strain
could be increased up to 1.2 g/L by changing the alcohol dehydroge-
nase gene from adhE2 to adhE1 (Inui et al., 2008). In another ap-
proach, 2-keto acids, the metabolic intermediates in amino acid
biosynthetic pathway, were used as precursors to produce butanol
and isobutanol by overexpressing the 2-keto acid decarboxylases
and alcohol dehydrogenases in E. coli (Atsumi et al., 2008b). When
the ilvD gene was additionally disrupted, 0.7 g/L butanol could be
produced by supplementing 8 g/L of L-threonine. To construct isobu-
tanol producer, 2-ketoisovalerate biosynthesis was additionally en-
hanced by disrupting the adhE, ldhA, frdAB, fnr, pta, pB genes
5 Y.-S. Jang et al. / Biotechnology Advances xxx (2011) xxxxxx
Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
while the alsS (Bacillus subtilis) and ilvCD (E. coli) genes were over-
expressed in E. coli strain. This metabolic engineering strategy led to
the production of 22 g/L isobutanol by culturing E. coli in a 250-mL
screw-cap conical ask (Atsumi et al., 2008b). In a very recent re-
port on application of metabolic engineering approaches for biobu-
tanol production in E. coli, construction of a chimeric pathway by
assembling the required genes from three different organisms has
been reported (Bond-Watts et al., 2011). For this purpose, the com-
bination of phaA (Ralstonia eutropha), hbd, crt, and adhE2 (C. aceto-
butylicum), and ter (Treponema denticola) genes was introduced into
the host E. coli. Additionally, the native aceEF-lpd operon of E. coli
was also overexpressed in order to provide reducing equivalents.
The metabolically engineered E. coli strain could produce a maxi-
mum of 4.7 g/L butanol in a 250-mL bafed ask sealed with paraf-
ilm (Bond-Watts et al., 2011).
2.4. Gasoline and diesel producers
Alkanes composed of 5 to 9 carbons, which are liquid at room
temperature, can be used as a good fuel in internal combustion en-
gine (Peralta-Yahya and Keasling, 2010). Alkanes composed of 10 to
16 carbons are more suitable for their use as diesel and aviation
fuel. Some eukaryotes naturally produce alkanes. For examples,
plants synthesize wax, which is an alkane consisting of more than
30 carbon atoms, to prevent evaporation of water, while insects pro-
duce pheromones are mainly hydrocarbons. Furthermore, biosyn-
thesis of alkanes (heptadecane as the most abundant one) has also
been reported in a diversity of microorganisms including photosyn-
thetic cyanobacteria (Dembitsky and Srebnik, 2002; Winters et al.,
Recently, an alkane-producing E. coli was developed by the intro-
duction of the alkane operon from cyanobacteria (Schirmer et al.,
2010). During this study, initially the genes responsible for alkane
biosynthesis in Synechococcus elongatus PCC7942 (orf1594 encoding
an acyl-acyl carrier protein reductase and orf1593 encoding an alde-
hyde decarbonylase) were identied and characterized. Subsequent-
ly, both orf1593 and orf1594 from S. elongatus were coexpressed in
E. coli. The resulting metabolically engineered E. coli strain could pro-
duce a mixture of alkanes (~ 0.3 g/L) including tridecane, pentade-
cene, pentadecane, and heptadecene at the approximate ratio of
10:10:40:40 (Schirmer et al., 2010).
Biodiesel could also be produced by E. coli after metabolic engi-
neering and synthetic biology for introducing new biochemical reac-
tion (Fig. 2C) (Steen et al., 2010). The metabolically engineered
E. coli strain could produce biodiesel through the acyltransferase ac-
tivity from acyl-CoA and ethanol, which are produced from the mod-
ied fatty acid biosynthetic pathway and the ethanol producing
pathways from Z. mobilis, respectively (Fig. 2C). The fatty acid biosyn-
thetic pathway of E. coli was modied to increase production of free
fatty acids and acyl-CoAs by disrupting the fadE gene encoding the
rst enzyme involved in beta-oxidation, and by overexpressing
genes for thioesterase and acyl-CoA ligase (Fig. 2C). To establish the
efcient biosynthetic pathway for ethanol production in E. coli, the
Z. mobilis pdc and adhB genes encoding pyruvate decarboxylase and
alcohol dehydrogenase, respectively, were introduced (Fig. 2C). The
metabolically engineered E. coli strain could produce fatty acid ethyl
esters (biodiesel) up to 674 mg/L, which corresponds to 9.4% of the
theoretical yield (Steen et al., 2010). When the genes encoding
endoxylanase catalytic domain from Clostridium stercorarium and a
xylanase from Bacteroides ovatus were introduced, of the engineered
E. coli could produce 11.6 mg/L of biodiesel from 2% (w/v) xylan
(Steen et al., 2010).
Through this approach, biogasoline and biodiesel, which are non-
native products, could be produced by engineered E. coli. Further-
more, systems metabolic engineering approach will become more
and more important for the development of strains capable of ef-
ciently producing biofuels and other products of interest.
2.5. Algal fuel and hydrogen
Photosynthetic algae have also been considered either as a bio-
mass source or host strain for the production of biofuel (Schmidt
et al., 2010). Some microalgae produce good raw materials, such as
triacylglycerol and starch, for their further conversion to biofuels
such as biodiesel and ethanol, or to hydrogen (Fig. 2D).
Hydrogen has long been considered as a good energy due to its
high energy density and sustainability. Hydrogen has been mainly
produced by algae and cyanobacteria, but at rather low efciencies.
Either hydrogenase or nitrogenase catalyzes hydrogen production
in photosynthetic algae and cyanobacteria (Benemann and Weare,
1974; Gaffron and Rubin, 1942). A highly efcient hydrogen produc-
er, Chlamydomonas reinhardtii Stm6, was isolated by random screen-
ing with the goal of increasing H
and e

supply to the
hydrogenase (Kruse et al., 2005). The resulting strain was able to pro-
duce hydrogen with 5 times higher yield than its parent strain. Met-
abolic engineering studies on the Smt6 strain, including the
introduction of a hexose symporter system from Chlorella kessleri
(Doebbe et al., 2007) and manipulation of the expression rates of
light harvesting protein subunits (Beckmann et al., 2009), were per-
formed to efciently produce hydrogen by water photolysis and to
enhance light capture efciency, respectively. The metabolically engi-
neered C. reinhardtii Smt6 strain showed 2030% higher growth rates
at high light (800 mE) condition and 1.5 times higher hydrogen pro-
duction rate compared to its parent strain (Beckmann et al., 2009).
In recent years, algal biodiesel has also attracted worldwide atten-
tion as carbon substrate other than carbon dioxide does not need to
be fed for fuel production. Various algal species produce intracellular
triacylglycerides to relatively high concentrations (Fig. 2D), which
vary from 20 to 77% of dry cell weight, depending on algal species.
Some of the best oil producing algal species include Botryococcus
braunii, Nannochloropsis sp., Schizochytrium sp. in which the triglycer-
ides content can vary between 2575%, 3168%, and 5077% of their
dry cell weights, respectively (Chisti, 2007; Hu et al., 2008). The tri-
glycerides produced by these algal species are subsequently used for
biodiesel production by trans-esterication process. Based on the bio-
chemical studies on fatty acid production, silicon deciency was
found to result in the accumulation of neutral lipids by increased ac-
tivity of acetyl-CoA carboxylase (ACCase) in Cyclotella cryptica
(Roessler, 1988). This ACCase catalyzes the conversion of acetyl-CoA
to malonyl-CoA, which is used as the substrate for fatty acid synthase
in this organism(Roessler, 1988). Based on this nding, metabolic en-
gineering of C. cryptica was attempted to enhance the expression
levels of the ACCase and UDP-glucose pyrophosphorylase (UGPase)
genes, which are important for lipid biosynthesis. However, lipid pro-
duction could not be enhanced despite of the overexpression of these
enzymes (Sheehan et al., 1998). Several attempts to improve triglyc-
eride production by metabolic engineering of green algae and dia-
toms did not result in much successful outcomes (Roessler, 1988,
1990; Roessler and Ohlrogge, 1993; Sheehan et al., 1998). More stud-
ies are needed to more systematically perform metabolic engineering
after improving our understanding on these organisms with respect
to genetics and physiology.
Ethanol is another class of liquid fuel that can be produced by
cyanobacteria by direct conversion of CO
via their inherent pho-
toautotrophic metabolism. Introduction of the Z. mobilis pdc and
adhII genes encoding pyruvate decarboxylase and alcohol dehy-
drogenase II into cyanobacteria resulted in ethanol production
(Deng and Coleman, 1999) (Fig. 2D). Recently, an ethanol produc-
ing Synechocystis strain has been developed by the chromosomal
integration of the Z. mobilis pdc and adhII genes (Dexter and Fu,
2009). While grown in photobioreactor, This metabolically
6 Y.-S. Jang et al. / Biotechnology Advances xxx (2011) xxxxxx
Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
engineered Synechocystis PCC 6803 strain could produce a maxi-
mum of 0.2 g ethanol/OD
unit/L/Day (Dexter and Fu, 2009).
The limited success on improving the performance of algal species
either by random and targeted mutagenesis or metabolic engineering
suggests that more rational and systematic approach needs to be ap-
plied for development of an efcient biofuel producing algal strain.
Thus, systems metabolic engineering needs to play its role to fully ex-
ploit the potential of biofuel production by algae. Several nuclear ge-
nome sequencing projects have been currently completed (http://, which will allow us to better un-
derstand genome-wide metabolic and regulatory characteristics and
provide us with the opportunity to redesign the metabolic pathway
systematically as done in many bacteria. In the near future, it is
expected that algal strains with improved capability of producing al-
cohols, diesels, alkanes, and hydrogen will be developed by systems
metabolic engineering concepts.
2.6. Isoprenoid-based fuels
Isoprenoids are derived from combination of two isomeric me-
tabolites, isoprenyl pyrophosphate (IPP) and dimethylallyl pyro-
phosphate (DMAPP), which are synthesized through two different
pathways, namely mevalonate (MEV) pathway and 1-deoxy-D-
xylulose 5-phosphate (DXP) pathway (Kirby and Keasling, 2008;
Muntendam et al., 2009; Peralta-Yahya and Keasling, 2010; Zhang
et al., 2011). Combination of IPP and DMAPP produces geranyl py-
rophosphate (GPP, C10), followed by farnesyl pyrophosphate
(FPP, C15), and geranylgeranyl pyrophosphate (GGPP, C20) via pre-
nyltransferases (Fig. 2E). These isoprenoids can be directed to two
different kinds of molecules that can be used as biofuels, depending
on the different enzymes utilized. One class of the corresponding en-
zymes is terpene synthases, which convert isoprenoids to cyclic al-
kenes or branched chains, while pyrophosphatase, the other
enzyme type, hydrolyzes isoprenoids to produce alcohols (Fig. 2E).
These metabolic pathways are of great importance in biofuels de-
velopment because all these metabolic products, including cyclic
alkenes (monoterpenes and diterpenes), branched alkanes (sesqui-
terpenes) and alcohols (farnesol, geraniol, and isopentenol) have
been proposed as gasoline substitutes, while long carbon chains
are better suited for diesel and jet fuel substitutes (Fig. 2E). Recent-
ly, E. coli has been engineered to enhance the production of farne-
sol (Wang et al., 2010a). Introduction of the foreign mevalonate
(MVA) pathway and overexpression of native FPP synthase in E.
coli have resulted in the production of 135.5 mg/L farnesol, while
no production of farnesol has been observed in the parent strain.
Recently, several metabolic engineering strategies have been
employed to overproduce isoprenoids in different hosts, including
plants and microorganisms, which have been well reviewed in
the recent papers (Kirby and Keasling, 2008; Muntendam et al.,
2009). The ideal concept to overproduce isoprenoids is the complete
transfer of a plant terpenoid pathway to microorganisms. Then, the
biosynthetic pathways need to be optimized in the engineered mi-
croorganisms. However, the regulation of the terpenoid pathway is
not simple, without mentioning the already complex networks of
the microbial host itself. Both heterologous and homologous net-
works can be properly regulated for enhancing the production of iso-
prenoids by systems metabolic engineering approaches.
3. Future perspectives on systems metabolic engineering for
biofuel production
To manufacture microbial strains for biofuel production, random
mutagenesis and metabolic engineering have been employed as stan-
dard strategies over the last couple of decades. However, the ap-
proach of random mutation and selection is difcult for further
improvement of cellular performance due to the complexity
associated with identifying modied gene as a consequence of ran-
dom mutation. Metabolic engineering, on the contrary, aims at im-
proving cellular performance by considering metabolic pathway in
entirety and manipulating specic genes that are targeted based on
engineering tools, which improved our knowledge of cellular physiol-
ogy and our subsequent engineering results (Lee et al., 2011a; Park and
Lee, 2008). In recent years, however, additional aspects of strain im-
provement had to be further considered for successful biochemical pro-
duction. High-throughput technologies and parallel advances of
computational and systems biology have enabled analyzing large
amount of omics data for investigating cellular metabolism and physiol-
ogy at systems-level. Also, engineering factors associated with up- and
down-stream processes have to be carefully examined. Metabolic engi-
neering integrated with systems-level analyses and computational
tools for the issues beyond simply engineering cells is termed systems
metabolic engineering, and is providing a newparadigmon the develop-
ment of strains for biofuel production (Lee et al., 2005a,2005b; Palsson
and Zengler, 2010; Park et al., 2008).
3.1. Tools and approaches employed systems metabolic engineering
Cellular functions can be mostly characterized by interrogating
three major networks in the cell: metabolic, gene regulatory and sig-
naling networks. Various tools have been developed, inexpensive yet
very fast genome sequencing, genome-wide proling of transcrip-
tome and proteome, DNA synthesis, and genome-scale metabolic net-
work simulation (Fig. 3). Having complete genome sequences and
annotations for the organisms of interest, comparative genome anal-
ysis is possible to identify the genes or regulatory regions that need
to be introduced, deleted, down- or up-regulated to attain a desired
metabolic phenotype. Accordingly, a strain to be engineered can be
compared with the genomes of other strains that contain interesting
metabolic and cellular phenotypes to identify the genes and regions
in the genome to be engineered (Lee et al., 2005a).
Transcriptome proling allows examination of the genome-wide
expression levels of mRNAs that can vary with genetic and environ-
mental conditions using DNA microarrays. Based on transcriptome
proling, potential target genes to be manipulated and strategies for
strain improvement can be identied by comparing the expression
levels of genes between the samples of same strain cultured under
different environmental conditions or between strains of different ge-
notypes under identical or different environmental conditions (Hibi
et al., 2007; Sindelar and Wendisch, 2007).
Similarly, proteome proling allows examination of the levels of
proteins in a cell by using two-dimensional gel electrophoresis
(2DGE) or chromatography-coupled mass spectrometry. Comparative
analysis of proteome proles between different samples under genet-
ically or environmentally different conditions can be used to identify
those proteins showing altered expression levels and those proteins
which are post-translationally modied (Lee and Lee, 2010), and
has been employed for strain improvement (Aldor et al., 2005; Han
et al., 2001, 2003). Recently, genome, transcriptome, proteome, and
other large-scale data were combined for better understanding on
cells to elucidate the transcriptional unit architecture of E. coli (Cho
et al., 2009). By integrating the information on organizational compo-
nents, such as RNA polymerase (RNAP)-binding regions, RNAP-
guided transcript segment, transcript start sites, and open reading
frames, the transcriptional structure of genomes in E. coli was deci-
phered and found to be much more complex than previously thought
(Cho et al., 2009). More recently, RNA sequencing data presenting the
transcriptional state has been algorithmically integrated into a
genome-scale metabolic model of Clostridium thermocellum (Gowen
and Fong, 2010).
Metabolomics allows proling metabolites, which are substrates,
products, and intermediates of cellular metabolism, under desired
culture conditions by using chromatography coupled with mass
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Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
spectrometry and nuclear magnetic resonance (NMR). The proles of
metabolites represent the metabolic status of a cell and thus provide
information on the physiological changes under genetic and environ-
mental perturbations. Metabolic ux proles, which can be consid-
ered as one of the ultimate phenotypes of a cell, are related closely
with cellular metabolic performance (Liebeke et al., 2011; Wittmann
and Heinzle, 2001). Fluxomics quanties metabolic uxes and collec-
tively represent the metabolic characteristics of a cell under a given
condition. Two methods,
C-based ux analysis and constraints-
based ux analysis, have been used to estimate the metabolic uxes
and understand the physiological and metabolic states of a cell in
the conditions of interest (Feng et al., 2010; Kim et al., 2008a; Rossell
et al., 2011). The
C-based ux analysis uses an isotope labeled sub-
strate, usually
C-labeled glucose, to quantify
C distribution pat-
terns of metabolites with NMR, liquid chromatographymass
spectrometry (LCMS), or gas chromatographymass spectrometry
(GCMS) (Sauer, 2006; Zamboni and Sauer, 2009). The computation-
al model and optimization techniques are integrated with the mea-
C-pattern data and exchange ux data, such as substrate
uptake rates and product excretion rates. Then, the intracellular met-
abolic uxes are estimated by minimizing the differences between
the simulated uxes and the experimentally measured data.
Fig. 3. Future perspectives for biofuel production using systems metabolic engineering and overall schematic procedure for strain development and application of the developed
strain. (A) Systems metabolic engineering strategy for the development of biofuel producers by integrating high-throughput experimental data with systematic analyses based
in silico genome-scale metabolic model and its simulations. (B) Overall strategy and procedure for strain development by systems metabolic engineering. Traditional methods to
develop improved strains, such as random mutagenesis or targeted metabolic engineering of selected genes, have limitations in dramatically improving the performance of a strain.
Systems biology allows us to investigate the organism at the systems-level using various genome-wide tools, including high-throughput analytical techniques, computational an-
alyses, and omics analyses that cover genome, transcriptome, proteome, metabolome, and uxome. Information obtained from such studies can be applied in an integrated manner
during the strain development by metabolic engineering. The whole process usually needs iteration until the desired phenotype and performance are obtained.
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Please cite this article as: Jang Y-S, et al, Engineering of microorganisms for the production of biofuels and perspectives based on systems
metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
C-based ux analysis calculates intracellular metabolic
uxes accurately, the scale of model is limited to rather small-scale.
Constraints-based ux analysis is based on an optimization technique
that calculates uxes by maximizing or minimizing one or more ob-
jective functions while satisfying the mass balances of metabolites
and stoichiometry of metabolic reactions under pseudo-steady state
assumption (Orth et al., 2010; Park et al., 2009). The methods of
constraints-based ux analysis can be applied to genome-scale meta-
bolic models. The in silico genome-scale metabolic models have been
reconstructed for many bacteria, archaea, and eukarya and more in
progress (Duarte et al., 2007; Durot et al., 2009; Feist and Palsson,
2008; Kim et al., 2008b; Schellenberger et al., 2010; Senger, 2010;
Thiele and Palsson, 2010). Also, several in silico algorithms have
been developed to predict the cellular physiology more accurately
(e.g. addition of physiological constraints) and identify target genes
to be deleted, introduced, and up-/down- regulated. The ux solution
space of in silico genome-scale metabolic model that represents the
all of feasible states of a cell is larger than physiologically possible
and feasible ux solution space of the real cell because the real cell
operates under various levels of cellular regulatory mechanisms,
such as transcriptional regulation, translational regulation, and ho-
meostasis. Thus, the addition of physiological constraints, represent-
ing experimental ux data (Sauer, 2006), transcriptional regulation
(Covert et al., 2008), thermodynamics (Henry et al., 2007), and phys-
iological characteristics of a cell (Beg et al., 2007; Park et al., 2010),
into the model, can improve the accuracy of predictions by reducing
the scope of broad ux solution space of a model (Park et al., 2009).
To identify target genes to manipulate for the overproduction of the
desired product, several in silico algorithms and strategies have
been introduced, including minimization of metabolic adjustment
(MOMA) (Segre et al., 2002), OptKnock (Burgard et al., 2003), OptReg
(Pharkya and Maranas, 2006), OptForce (Ranganathan et al., 2010),
and ux scanning based on enforced objective ux (FSEOF) (Choi
et al., 2010). Furthermore, in silico genome-scale modeling and simu-
lation can incorporate several omics data to promote our capability
for understanding the metabolic characteristics of a cell under any ge-
netic and environmental perturbations, and then develop metabolic
engineering strategies for strain improvement at systems-level.
3.2. Production of biofuels using systems metabolic engineering
Systems metabolic engineering allows systematic changes of met-
abolic pathways toward desired goals including enhancement of
product concentration, yield and productivity. Accordingly, there
have recently been several reports on the use of systems metabolic
engineering in developing microbial hosts for the production of bio-
fuels (Alsaker et al., 2010; Bro et al., 2006; Hjersted et al., 2007; Kim
and Reed, 2010; Ranganathan and Maranas, 2010). Some of these ex-
amples are described below, which are also summarized in Table 1.
Systems metabolic engineering can provide novel solutions and
strategies for further enhancing ethanol production. Recently, in silico
genome-scale metabolic models for ethanologenic organisms, such as
E. coli (Feist and Palsson, 2008), Z. mobilis (Lee et al., 2010), and S. cer-
evisiae (Mo et al., 2009), have been developed. In silico genome-scale
metabolic models are powerful and promising tools to explore cellu-
lar characteristics at systems-level and design metabolic engineering
strategies for the improved and desired properties of a cell (Blazeck
and Alper, 2010; Kimet al., 2008b, 2011; Milne et al., 2009; Oberhardt
et al., 2009). For example, the genome-scale metabolic model of S.
cerevisiae aided the improvement of ethanol production to increase
ethanol yield and decrease glycerol production under anaerobic con-
ditions using glucose as a carbon source (Bro et al., 2006; Hjersted
et al., 2007). OptReg method suggests the optimal metabolic engi-
neering strategies of activation, repression, and elimination of reac-
tions for the overproduction of ethanol in E. coli (Pharkya and
Maranas, 2006). Also, OptORF method was developed to identify
optimal gene knockout and amplication targets for strain improve-
ment by considering transcriptional regulatory network in addition
to metabolic network, and employed for the production of ethanol
and higher alcohols including isobutanol in E. coli (Kim and Reed,
2010). One approach to identify target genes to be manipulated for
increasing tolerance against ethanol is transcriptome analysis. In-
creased expression of genes involved in glycine and betaine degrada-
tion showed benecial effects on ethanol tolerance (Gonzalez et al.,
2003). Addition of glycine and betaine increased ethanol tolerance
by over 2-fold in engineered E. coli strain (Gonzalez et al., 2003).
More examples of applying genome-scale metabolic simulation and
analysis on the development of ethanol producers are expected.
In the case of the butanol production, after the genome sequenc-
ing of clostridia, more systematic approaches became available for
the metabolic engineering. The complete genome sequences of C.
acetobutylicum (Nolling et al., 2001) and C. beijerinckii NCIMB 8052
( have been reported.
More recently, in silico genome-scale metabolic models have been
reconstructed by two independent research groups (Lee et al.,
2008a; Senger and Papoutsakis, 2008a,2008b), which will allow
genome-scale ux analysis and other simulations for designing the
metabolic engineering strategies. In silico simulation methods devel-
oped for identifying engineering targets were also applied for the pro-
duction of butanol. The method called OptForce examines possible
ux ranges for all metabolic reactions in bioproduct-overproducing
networks compared with the wild type metabolic network, and se-
lects genes to be amplied, repressed, and eliminated. This strategy
was implemented to investigate the metabolic network for the in-
creased production of butanol in E. coli (Ranganathan et al., 2010;
Ranganathan and Maranas, 2010). Additionally, in silico approaches
in E. coli were applied for the high level production of L-valine and
L-threonine (Lee et al., 2007; Park et al., 2007, 2011) whose biosyn-
thetic pathways could be used for the production of butanol and
higher alcohols (Atsumi et al., 2008b; Shen and Liao, 2008). In other
studies, computational algorithms have been suggested to predict
metabolic pathways feasible for the production of the target chemi-
cals, including butanol, on the basis of chemical structure changes,
enzymatic information, thermodynamic constraints, and reaction
mechanisms (Cho et al., 2010; Li et al., 2004). These powerful tools
for the prediction of pathways will greatly contribute constructing ar-
ticial metabolic pathways for more efcient production of butanol
and other biofuels.
Based on the complete genome sequence, genome-wide transcrip-
tome (Tomas et al., 2003b, 2004) and proteome studies on C. acetobu-
tylicum (Alsaker et al., 2010; Mao et al., 2010; Schwarz et al., 2007;
Sullivan and Bennett, 2006) have been performed. Microarray exper-
iments have been used to investigate stress responses by several me-
tabolites, such as butanol, butyrate, and acetate (Alsaker et al., 2010),
and sporulation of C. acetobutylicum (Alsaker et al., 2004; Jones et al.,
2008; Tomas et al., 2003a). Indeed, investigation on the responses to
solvent stress in C. acetobutylicum by transcriptome analysis identi-
ed molecular chaperones including groES, dnaKJ, hsp18, and hsp90
as potential target genes for improving solvent tolerance coupled
with genes involved in sporulation, fatty acid synthesis and transcrip-
tional regulators (Tomas et al., 2004). Genomic library screening of
C. acetobutylicum (Borden and Papoutsakis, 2007) and E. coli (Reyes
et al., 2011) have also been conducted to improve butanol tolerance
in a genome-wide manner. In these studies, CAC0003 and CAC1869
genes in C. acetobutylicum (Borden and Papoutsakis, 2007) and the
entC and feoA genes in E. coli (Reyes et al., 2011) were identied to in-
crease butanol tolerance. Furthermore, it was recently reported that
non-coding RNAs (ncRNAs) might affect metabolite tolerance in
C. acetobutylicum (Borden et al., 2010); the ncRNA named RDNA7 in-
creased butyrate tolerance of C. acetobutylicum.
Genome-wide systems analyses were also applied to the produc-
tion studies of isobutanol. Whole genome sequencing followed by
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metabolic engineering approaches, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.015
gene repair and knockout identied several genes (acrA, gatY, tnaA,
yhbJ, and marCRAB) involved in isobutanol tolerance of E. coli (Atsumi
et al., 2011). More recently, evolution combined with genomic study
identied that the marC, hfq, mdh, acrAB, gatYZABCD and rph genes led
to increased tolerance to isobutanol stress in E. coli (Minty et al.,
Consequently, systems metabolic engineering for biofuel produc-
tion will allow us to comprehend the cellular physiology more accu-
rately, expand the understandable scope of engineering, and
produce newinformation on the biological systems of cell. Employing
the systems metabolic engineering for biofuel production is expected
to improve the attainable performance of cell and become a standard
approach for worldwide fuel production using microbial platforms
(Fig. 3).
4. Conclusions
Limited fossil oil resources and increasing environmental concerns
are urging us to establish biorenery systems for sustainable and eco-
nomical production of alternative fuels. In order to develop econom-
ical and sustainable processes for biofuel production, the metabolic
pathways of biofuel producers need to be optimally redesigned to
achieve high performance. The major goals of metabolic pathway
redesigning for biofuel producer include improved product yield,
higher product concentration and productivity, and product toler-
ance. Also, the production strain should be designed in a way that
the whole process becomes operationally inexpensive by system-
wide optimization of midstream and downstream processes. We are
ready for taking a second leap toward efcient strain development
by systems metabolic engineering that integrates traditional meta-
bolic engineering and bioprocess engineering with systems biology
and synthetic biology. It is expected that this combined strategy will
result in the development of microorganisms capable of producing
various biofuels cost effectively on industrial scale.
We thank Dr. Jin Hwan Park for his valuable comments to the
manuscript. This work was supported by the Advanced Biomass
R&D Center of Korea (ABC-2010-0029799) through the Global Fron-
tier Research Program of the Ministry of Education, Science and Tech-
nology (MEST). Further support by the GS Caltex, BioFuelChem, EEWS
program of KAIST, and the World Class University program (R32-
2008-000-10142-0) of the MEST are appreciated.
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