Metabolic Engineering of E. Coli. A Sustainable Industrial Platform For Bio-Based Chemical Production

Biotechnology Advances 31 (2013) 12001223
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Metabolic engineering of Escherichia coli: A sustainable industrial

platform for bio-based chemical production
Xianzhong Chen a,, Li Zhou a, Kangming Tian a, Ashwani Kumar b, Suren Singh b,
Bernard A. Prior c, Zhengxiang Wang d,
a
Key Laboratory of Industrial Biotechnology, Ministry of Education & School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
Department of Biotechnology & Food Technology, Faculty of Applied Sciences, Durban University of Technology, P.O. Box 1334, Durban, 4001, South Africa
Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa
d
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education & The College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
b
c
a r t i c l e
i n f o
Article history:
Received 18 October 2012
Received in revised form 4 February 2013
Accepted 25 February 2013
Available online 6 March 2013
Keywords:
Escherichia coli
Metabolic engineering
Bioprocess
Biochemical product
Industrial platform
a b s t r a c t
In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk
and/or ne chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a
fundamental and applied level and has become a predominant host microorganism for industrial applications.
Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been signicantly
promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary
engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a
sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids,
amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by
E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted.
Moreover, this review presents guidelines for future developments in the bio-based chemical production using
E. coli as an industrial platform.
2013 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biofuels production . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Hydrogen production . . . . . . . . . . . . . . . . . . . . .
2.2.
Bioethanol production . . . . . . . . . . . . . . . . . . . . .
2.3.
Advanced biofuels production using recombinant E. coli as efcient
2.3.1.
1-Butanol and 1-propanol production . . . . . . . . .
2.3.2.
2-Methyl-1-butanol and 3-methyl-1-butanol production
2.3.3.
Isopropanol and isobutanol production . . . . . . . . .
Organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Lactic acid production . . . . . . . . . . . . . . . . . . . . .
3.2.
Succinic acid production . . . . . . . . . . . . . . . . . . . .
3.3.
Production of other organic acids . . . . . . . . . . . . . . . .
Amino acids production . . . . . . . . . . . . . . . . . . . . . . .
L-Threonine production . . . . . . . . . . . . . . . . . . . .
4.1.
L-Valine production . . . . . . . . . . . . . . . . . . . . . .
4.2.
L-Phenylalanine production
. . . . . . . . . . . . . . . . . .
4.3.
4.4.
Production of other amino acids . . . . . . . . . . . . . . . .
Sugar alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel./fax: +86 510 85918122.

Corresponding author. Tel/fax: +86 022 60601958.
E-mail addresses: xzchen@jiangnan.edu.cn (X. Chen), zx.wang@tust.edu.cn (Z. Wang).
0734-9750/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.02.009
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X. Chen et al. / Biotechnology Advances 31 (2013) 12001223
6.
Biopolymer and monomers production . . . . . . .

6.1.
1,3-Propanediol and 1,2-propanediol . . . . .
6.2.
1,4-Butanediol and 2,3-butanediol . . . . . .
6.3.
Polyhydroxyalkanoates production . . . . . .
6.4.
Polylactic acid and copolymers production . .
7.
Biosynthesis of complex natural compounds in E. coli
7.1.
Isoprenoid biosynthesis . . . . . . . . . . .
7.2.
Nonribosomal peptides and polyketides . . .
7.3.
Coenzyme Q10 production . . . . . . . . .
8.
Conclusion and future directions . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Currently commodity chemicals and traditional fuels are predominantly produced from fossil based resources and have played an
essential role in international social and economic development.
However, with the rising concerns of the sustainability, carbon dioxide
release into the atmosphere and the negatively environmental impact
of traditional petrochemical based products and fossil fuels, the biotechnological route of production from renewable carbon sources is
a desirable alternative to petrochemical-based production (Bozell
and Petersen, 2010; Bruschi et al., 2011). The economic potential of
biotechnology industry has become an important contribution to the
world economy in recent years: its global value is estimated about
500 billion dollars in 2011, compared to only 54 billion dollars in
1999 and 101 billion dollars in 2003 (Bruschi et al., 2011). Increasingly
bulk chemicals (such as organic acid, amino acid, and biofuels) and ne
chemicals (antibiotics, vitamins, pharmacy chemicals, sweeteners, and
performance materials) have been produced by the intervention of biotechnology at an industrial scale. Many industrial processes are based
on the catalytic activities of microorganisms and therefore strains
need to be developed and selected that grow rapidly to a high cell
density with high volumetric and specic productivities (activity per
fermentation volume or biomass), and yield products from substrates
close to the theoretical maximum. Additional properties of high stress
tolerance, and simple molecular manipulation based on available genetic
information are desirable (Huffer et al., 2012).
Extensive fundamental studies on Escherichia coli over the last
50 years have resulted in the bacterium becoming the prime prokaryotic genetic model. Moreover, since the beginning of the modern
biotechnology era in the late 70s, E. coli has been used widely for molecular cloning methodologies and as a host to produce primary and
secondary metabolites. Representative biochemical products are
summarized in Table 1. E. coli possesses a number of excellent properties, such as a rapid doubling time and growth rate, ease of high-celldensity fermentation, low production cost and, detailed knowledge of
the metabolism and most importantly, the availability of excellent
genetic tools for strain improvement. These characteristics make
E. coli an ideal candidate for both metabolic engineering and
commercial-scale production of desirable bioproducts (Table 1).
Traditionally, strain improvement was achieved mainly by multiple
rounds of random mutagenesis and selection, which are still very useful
nowadays (Portnoy et al., 2011; Sonderegger and Sauer, 2003). In the
latest decade, the development of gene deletion approaches (Bloor
and Cranenburgh, 2006; Datsenko and Wanner, 2000; Murphy et al.,
2000) enabled efcient genome DNA inactivation and greatly improved
metabolic engineering of E. coli. A more systematic and integrated
approach for biotechnological process development and optimization
became prevalent. Furthermore, metabolic engineering of E. coli has
been employed to broaden the variety of available products (Table 1).
In view of the above, this review highlights the current trends towards
the bio-based production of chemicals using genetically modied E. coli
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1201
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1213
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as a sustainable biocatalytic platform and the impact on industrial biotechnology. Therefore, essential primary and secondary metabolites
produced by engineered E. coli are evaluated and discussed. In addition
we show that systematically engineered E. coli will pave a broad avenue
for the development of a green replacement for petrochemical products
and play a critical role as a novel platform in industrial microorganism
and pharmaceutical biotechnology. The physiology, biochemistry,
genomics and methodology of this microorganism have been extensively studied (Blattner et al., 1997; Bloor and Cranenburgh, 2006)
and are outside the scope of this review whereas strategies to improve
the catalytic efciency of E. coli are given more emphasis.
2. Biofuels production
Concerning availability and abundance of biomass resources, as
well as environmental pollution of fossil fuels, development of
bioenergy as an alternative fuels has gained more attention in recent
past. Generally, biofuels includes the hydrogen, bioethanol and advanced
fuels (Table 1) but the catabolic pathways to synthesize these products
are diverse (Fig. 1). In this section, we discuss the progress in biofuels
production by engineered E. coli and highlight some of successful strategies for increasing catalytic efciency of cell.
2.1. Hydrogen production
As an alternative for petroleum fuels, microbial production of
hydrogen as a future fuel is a promising possibility (Kim and Lee, 2010;
Panagiotopoulos et al., 2009). Hydrogen is a highly energy dense source
and its conversion to heat or power is simple and clean when combusted
with oxygen as only water is formed without generation of pollutants
(Hoffmann, 2002). Biological production of hydrogen using biomass as
a feedstock is less energy-intensive, sustainable, eco-friendly, and considered to be neutral for CO2 emissions (Panagiotopoulos et al., 2009).
A number of newly isolated microbes i.e. Thermoanaerobacterium
thermosaccharolyticum, Clostridium beijerinckii and Sporoacetigenium
mesophilum have been reported in literature for hydrogen production,
but with limited production (Cai et al., 2011; Oh et al., 2011). The low
yield has been a major obstacle for the hydrogen production through
microbial fermentation of glucose. Kim et al. (2009) attempted to overcome the limitations with natural isolates by metabolically engineering
E. coli strains for hydrogen production. Deletion of hycA, a negative
regulator for formate hydrogen lyase and two uptake hydrogenases
(hya and hyb), resulted in carbon ux alterations to H2 pathway. H2
yield was further improved to 2.11 mol/mol glucose fermented by
deletion of lactate dehydrogenase (ldhA) and fumarate reductase
(frdAB) under reduced H2 pressure in batch experiments. Using a similar
genetic approach, H2 was produced with an improved yield by an
engineered E. coli strain (Maeda et al., 2008). A recent report showed a
high volumetric productivity of 2.4 H2/L/h using immobilized cells of a
genetically recombinant E. coli, which has deletion mutations in uptake
hydrogenases (hyaAB), lactate dehydrogenase (ldhA) and fumarate
1202
Table 1
Representative bio-based chemicals produced by genetically engineered E. coli.
Bio-based chemical
Industrial
applications
Engineering strategy
Fermentation process
Carbon
source
Titer and yield
Refs
Biofuels
Hydrogen
Fuel and energy carrier
Immobilized
recombinant cells
Formate
Fuel, solvent, food,

beverage
10-L bioreactor batch

anaerobic cultivation
Xylose and
glucose
1-Propanol
Gasoline additive,
allround solvent
Shake asks and IPTG

induction
Glucose
1.0 mol H2/mol

formate; 2.4 l
H2/L/ha; formate.
38.81 g/L;
0.49 g/g glucose
or xylose
3.5 g/L;
0.049 g/gb
Seol et al.
(2011)
Bioethanol
1-Butanol
Bulk material, gasoline

additive or fuel
30 g/L; 88% of the

theoretical yield
Shen et al.
(2011)
Advanced fuel,
alternative gasoline
Glucose
9.5 g/L; 0.11 g/g

glucose
Connor et al.,
(2010)
Isopropanol
Biodiesel, precursor of
polypropylene
Glucose
143 g/L; 67.4%

(mol/mol)
Inokuma et
al. (2010)
Isobutanol
Gasoline blend stock,

precursor of butenes
1-L Bioreactor with

aerobicanaerobic dual
phase fermentation
Shake ask with
two-phase fermentation
to remove product
Fed-batch fermentation,
gas-stripping-based
recovery process
Screw-cap conical asks
and IPTG induction
Glucose
3-Methyl1-butanol
Glucose
20 g/L; 86% of the

theoretical
maximum
Atsumi et al.
(2008b)
L-Lactic
Food, beverages,
biopolymer
Deletion of negative regulator and

competing carbon metabolic
pathways
Minimized metabolic functionality for
conversion of xylose and glucose into
ethanol by multiple-gene knockout
Improving specic activity and
releasing feedback inhibition of the
key enzyme by directed evolution
Construction of modied clostridial
1-butanol pathway in E. coli strain
and enhancement of driving forces
Random mutagenesis and selection
combined with overexpression of key
genes
Heterologous expression of target
product pathway from various
sources in E. coli host
Introducing non-fermentative
synthetic pathway in E. coli and
elimination of pathways competing
for pyruvate and cofactors
Overexpression of L-lactate
dehydrogenase and deletion of genes
responsible for by-products pathway
Disruption of the competing
pathways and expression of key
enzyme
Inactivation of ptsG, p and ldhA,
combined overexpression of pyc gene
Glucose and
xylose
57.1 g/L; 0.83 g/g

of total sugar
Dien et al.
(2002)
Glucose
122.8 g/L;
0.866 g/g
Zhou et al.
(2011b)
Glucose
99.2 g/L; 110%
Vemuri et al.
(2002)
Glucose
90 g/L; 0.68 g/g
Glucose
878 mM; 75% of

the theoretical
maximum
82.4 g/L;
0.393 g/g
Zhu et al.
(2008)
Causey et al.
(2003)
acid
500-mL Flask with

automatic pH control,
batch fermentation
D-Lactic acid
Cosmetics,
7-l Bioreactor aerobic and
pharmaceuticals
oxygen-limited fed-batch
biopolymer
fermentation
Succinic acid
Bulk chemical, food,
2.5-L Fermentor
agriculture
aerobic-anaerobic
process fed-batch
Pyruvate
Food, pharmaceuticals,
Elevation of glycolytic ux to
2.5-L Fermentor
and chemicals
pyruvate by multiple-gene knockout fed-batch process
Acetate
Food, plastics, solvents, Genetic modication with blocking of 14-L Fermentor with
and de-icers
by-products pathways, oxidative
fed-batch process and DO
phosphorlation and TCA function
control
L-Threonine
Food, antibiotics,
System metabolic engineering with
6.6-L Fermentor pH-stat
Amino
pharmaceuticals, animal transcriptome proling and in silico
fed-batch culture
acids
feed
ux response analysis
L-Valine
Cosmetics,
Systematically metabolic engineering Batch fermentation in
pharmaceuticals, feed
with transcriptome analysis and in
shake asks
additive
silico genome-scale simulation
L-Phenylalanine Food industry,
Reverse engineering with metabolic
Shake asks with IPTG
Sweetener aspartame
transcription analysis, deletion of PTS induction process
system and expression of key genes
L-Tryptophan
Food, animal feed,
Implementation of temperature3-L Fermentor with
pharmaceutical
inducible expression system, eliminafed-batch fermentation,
industries
tion of feedback inhibitions and degra- temperature-switching
dation pathway of desired product
method
L-Tyrosine
Precursor of drugs,
Expression of the key feedback
1-L Bioreactors batch
biopolymers, melanin
inhibition-insensitive enzyme from
fermentation
Z.mobilis
Sugar
Xylitol
Pharmaceutical and
Heterologous overexpression of the
Aerobic growth- anaeroalcoholos
food industry
key enzymes in xyltiol biosynthetic
bic production, fed-batch
pathway and construction of cofactor fermentation
regeneration
Mannitol
Pharmaceutical and
Construction of cofactor cycle system Whole-cell
food industry
and heterologous expression of the
biotransformation with
key enzymes
pH-static conditions
Diols and
1,3-PDO
Monomers for the
Synergetic introduction of glycerol
Fed-batch fermentation
polymers
synthesis of polyester
pathway from S. cerevisiae and
1,3-PDO pathway from K. pneumonia
1,2-PDO
Polyester, antifreeze,
Heterologous overexpression of the
Fermentor containing
and deicer
key enzymes and disruption of
500-mL medium, batch
undesired by-products pathways
anaerobic fermentation
1,4-BDO
Polymers, ne
Implementation of synthetic biology
2-L Bioreactors with
chemicals and solvents. and systematic metabolic engineering, aerobic-microaerobic
and rational metabolic engineering
two phase fed-batch
(R,R)-2,3-BDO
Polymers, food,
cosmetics, and
pharmaceuticals
Evaluation of various synthetic

operons consisting of the 2,3-BDO
biosynthesis pathway with different
secondary alcohol dehydrogenases
Shake asks with IPTG

induction process
Glucose
Trinh et al.
(2008)
Atsumi and
Liao (2008a)
Lee et al.
(2007)
Glucose
7.55 g/L;
0.378 g/g
Park et al.
(2007)
Glucose
0.33 g/g
Baez-Viveros
et al. (2007)
Glucose
13.3 g/L; 0.1 g/g
Zhao et al.
(2011)
Glucose
3 g/L; 66 mg/g
Chavez-Bejar
et al. (2008)
Glucose and
xylose
8.52 g/Lb;
4 mol/mol
Akinterinwa
and Cirino
(2011)
D-fructose
362 mM;
0.84 mol/mol
Kaup et al.
(2004)
Glucose
130 g/L
Emptage et
al. (2003)
Glycerol
5.6 g/L; 0.213 g/g
Either of
glucose,
xylose or
sucrose
Glucose
18 g/L
Clomburg
and Gonzalez
(2011)
Yim et al.
(2011)
6.1 g/L; 0.31 g/g
Yan et al.
(2009)
1203
Table 1 (continued)
Bio-based chemical
a
b
c
d
Industrial
applications
Engineering strategy
Fermentation process
Carbon
source
Titer and yield
Refs
PHA
Biopolyesters and
biofuel
141.6 g/L; 4.63 g

of PHB/L/h.c
Choi et al.
(1998)
Taxol precursor, a
potent anticancer drug
6.6-L Fermentor
fed-batch culture with
pH-control
Fed-batch and liquid
liquid two-phase cultivation carried out in 1-L
bioreactors
Glucose
Taxadiene
Glucose
1.02 g/L
Ajikumar et
al. (2010)
Echinomycin
Antitumor, antibacterial
and antiviral activity
Glucose
0.6 mg/L
Watanabe et
al. (2006)
Anthracyclines
Bacterial aromatic
polyketides, antibiotics
and anticancer drugs
3 mg/L
Zhang et al.
(2008)
CoQ10
Medicine, foods and

cosmetics
Constitutive expression of an operon

responsible for PHA biosynthesis
pathway of A. latus in E. coli
Synthetic biology and
implementation of the
multivariate-modular approach to
optimally balance the two pathway
modules
Cloning and heterologous expression
in E. coli of a monocistronic
reconstituted form of gene cluster
responsible for echinomycin
biosynthetic pathway
The total biosynthesis of
anthracyclines by dissection and
reassembly of fungal polyketide
synthase into a synthetic PKS
Improvement of IPP supply by introducing a foreign mevalonate pathway
and coexpression of decaprenyl diphosphate synthasegene and IPP
isomerase
2700 g/g DCWd
Zahiri et al.
(2006b)
Fed-batch fermentation
carried out in 1-L bioreactors and minimal
medium
High-cell density,
Glucose
fed-batch fermentation in
2-L fermentor, IPTG
induction
Fermentation with a
Glycerol
rotary shaking incubator
and optimization of pH
Proudction rate of H2.

Calculated based on the references data.
PHB productivity.
The result was obtained when supplemented with exogenous mevalonate of 3 mM.
dehydrogenase (frdAB) (Seol et al., 2011). Although, biological H2 production was signicantly improved by genetically modied E. coli strain,
many critical barriers involved in the yield, productivity and metabolic
robustness are still not at a level that would allow commercialization.
2.2. Bioethanol production
Bioethanol is the predominant renewable liquid energy source
capturing 90% of the current world biofuel market (Antoni et al.,
2007), with annual production of more than 105 billion liters in
2011. Production by Saccharomyces cerevisiae of starch-based ethanol
and sugar cane-based ethanol are now mature industries but these
biomass sources compete with food and feed (Geddes et al., 2011b),
which leads to a global increase in the price and demand for food
crops. Ethanol production from xylose, glucose or mixture sugars
(main compounds of plant lignocellulose residues) has been intensively
investigated for more than 20 years (Alterthum and Ingram, 1989; Ohta
et al., 1990) and many companies are attempting to commercialize the
process. The recalcitrance of lignocellulose (cellulose and hemicellulose) to direct microbial fermentation to ethanol has resulted in the
requirement for complicated pretreatment processes (Himmel et al.,
2007). Ethanologenic E. coli strains have the advantage that they are
able to ferment pretreated biomass directly (Alterthum and Ingram,
1989; Moniruzzaman et al., 1997; Zaldivar et al., 2000; Zheng et al.,
2012). For example, E. coli strains which are adapted to phosphoric
acid hydrolysates, can ferment hemicellulose and cellulose-derived
sugars together in a single 80-l bioreactor vessel, termed simultaneous
saccharication and co-fermentation (SScF), and an ethanol yield of
0.27 g/g bagasse was obtained (Nieves et al., 2011). In another example,
deletion of mgsA encoding methylglyoxal synthase (and methylglyoxal)
resulted in the co-metabolism of glucose and xylose, and increased
the fermentation rate of ethanologenic E. coli by accelerating the cometabolism of hexose and pentose sugars to ethanol (Yomano et al.,
2009).
However, inhibitors present in lignocellulose hydrolysates such as
furfural, 5-hydroxymethyl furfural, acetate and soluble products
released from dilute acid pretreatment are toxic for most organisms
including E. coli and reduce ethanol yield drastically (Mills et al.,
2009). The toxicity problem can be partially overcome by increasing
tolerance of strains to hydrolysate inhibitors. In recent reports,
some E. coli metabolic regulators involved in resistance to furfural

have been identied (Miller et al., 2009a, 2009b; Turner et al., 2011;
Wang et al., 2011c). Miller et al. (2009a, 2009b) found that silencing
of yqhD, an NADPH-dependent furfural oxidoreductase induced by
furfural, can result in resistance of E. coli cells to low concentrations
of furfural. Furthermore, the enzyme of YqhD has unusually low Km
values for NADPH that would allow to compete with biosynthesis for
NADPH, which appears to be the primary basis for growth inhibition
by furfural (Miller et al., 2009b). The combination of deletion of yqhD
and overexpression of fucO, an NADH-dependent, L-1,2-propanediol
reductase, can provide a further benet for furfural tolerance (Wang
et al., 2011c). In addition, Wang et al. (2012) found that plasmid expression of ucpA, a putative oxidoreductases gene, improved the furan tolerance in both the native W strain and ethanologenic strain LY180.
Deletion of the chromosomal ucpA decreased furfural tolerance, providing a clear phenotype for this cryptic gene.
On the other hand, hydrolysate resistant E. coli strains, which were
generated by either adaptive evolution or metabolic engineering
(Miller et al., 2009b; Mills et al., 2009; Wang et al., 2012), combined in
a SScF process resulted in both higher yields and less process complexity
(Geddes et al., 2011a). Accordingly, Ingram and coworkers (Wang et al.,
2011c) found that the overexpression of NADH-dependent propanediol
oxidoreductase can improve the furfural tolerance of E. coli cells, which
provided a new approach to improve furfural tolerance. In a recent example, Edwards et al. (2011) constructed a recombinant ethanologenic
E. coli strain by introducing the Klebsiella oxytoca cellobiose
phosphotransferase genes, a pectate lyase with secretion capability
and oligogalacturonide lyase gene from Erwinia chrysanthemi. This modied strain efciently converted polygalacturonate to monomeric sugars
and an increased ethanol production of 45% was achieved compared to
the control strain. In our studies, we have created a recombinant E. coli
strain, which can covert xylose to ethanol by introducing pdc and adhB
genes from Zymomonas mobilis (Sun et al., 2004). Synchronous expression of pdc and adhB genes encoding pyruvate decarboxylase and
alcohol dehydrogenase, respectively, redirected the carbon ux into
the ethanol production and the resulting recombinant strain produced
ethanol up to 1.28% (v/v) using xylose as carbon source within 36 h
fermentation. Similar approaches were implemented to engineer
E. coli for ethanol production from xylose, glucose or mixed sugars
(Sanny et al., 2010; Wang et al., 2008). Interestingly, E. coli with a
1204
Fermentative
pathways
1-Butanol
adc (Ca)
adh (Cb)
Acetoacetate
Acetone
Isopropanol
adhE2 (Ca)
atoAD (EC)
Butyraldehyde
ctfAB (Ca)
Ethanol
Acetate
adhE2 (Ca)
adhE (Ec)
atoB (Ec)
Butyryl-CoA
Acetyl-CoA
Acetoacetyl-CoA
adhB (Zm)
adhE (Ec)
Acetaldehyde
thl (Ca)
bcd-etfBA (Ca)
hbd (Ca)
crt (Ca)
Crotonyl-CoA
3-Hydroxybutyl-CoA
Pyruvate
pdc (Zm)
Glucose
1-Propanol
L-Threonine
Valine
biosynthesis
kivd (Ll)
adh2 (Sc)
2-Ketobutyrate
Norvaline
biosynthesis
2-Ketovalerate
Isoleucine
biosynthesis
2-Keto-3methyl-valerate
1-Butanol
2-Ketoisolvalerate
Leucine biosynthesis
Kivd (Ll)
adh2 (Sc)
2-Keto-4-methylpentanoate
Isobutanol
Kivd (Ll)
adh2 (Sc)
Pyruvate
Nonfermentative
pathways
Kivd (Ll)
Kivd (Ll)
adh2 (Sc)
adh2 (Sc)
2-Methyl-1-butanol
3-Methyl-1-butanol
Fig. 1. Fermentative pathways and nonfermentative pathways in E. coli for the production of a number of biofuels (Clomburg and Gonzalez, 2010; Shen et al., 2011). Various synthetic and metabolic strategies have been successfully employed for the enhanced production of candidate biofuel compounds (shown in the brown shaded boxes). Relevant reactions are represented by the name of the genes and enzymes. Abbreviations of genes and enzymes: adc, acetoacetate dehydrogenase; adh, secondary alcohol dehydrogenase;
adhB, alcohol dehydrogenase; adhE, acetaldehyde/alcohol dehydrogenase; adhE2, secondary alcohol dehydrogenase; atoAD, acetyl-CoA:acetoacetyl-CoA transferase; atoB,
acetyl-CoA acyltransferase; bcd, butyryl-CoA dehydrogenase; crt, crotonase; ctfAB, acetoacetyl-CoA transferase; etfBA, electrotransfer avor protein; hbd, -hydroxy butyryl-CoA dehydrogenase; pdc, pyruvate decarboxylase; thl, acetyl-CoA acyltransferase; kivd, ketoisovalerate decarboxylase; adh2, alcohol dehydrogenase; Cb, C. boidinii; Ca, C. acetobutylicum; Ec, E.
coli. Ll, L. lactis; Sc, S. cerevisiae.
minimal metabolic functionality was rationally designed and

constructed for ethanol production based on metabolic pathway analysis for cellular network (Trinh et al., 2008). With the disruption of
nonessential pathways, the strain produced 39 g/L ethanol through
simultaneous utilization of pentoses and hexoses with a highest yield
close to the theoretical maximum. This rational approach may be useful
for strain development to enhance different products. Conclusively,
E. coli, the workhorse of modern biotechnology, has become a promising
host for the microbial production of biofuels due to the ease of genetic
manipulation, high yields, capability of utilizing a wide variety of
substrates, and high growth and metabolic rates (Ingram et al., 1987;
Ohta et al., 1991; Wang et al., 2008). However, it should be noted that
compared to S. cerevisiae, its ability to produce high concentrations of
ethanol, and tolerance to ethanol and other inhibitory compounds of

lignocellulosic biomass is lower and not commercially viable for E. coli
strains, pointing to the need to employ systems metabolic engineering
for strain development in future.
In addition to glucose and xylose, E. coli is capable of utilizing glycerol.
These substrates are the most readily available renewable feedstock.
With the increasing international biodiesel production, a surplus of
crude glycerol has been generated resulting in a signicant price reduction. Value-added chemical production using glycerol as feedstock has
become attractive recently. In addition to low cost and abundance, the
higher degree of reduction compared to sugars such as glucose and
xylose makes glycerol be an excellent potential carbon source to produce
chemicals with high yield (Clomburg and Gonzalez, 2010). Previously, it
was thought that wild type E. coli cannot convert glycerol into ethanol in
chemically dened medium under anaerobic condition because of lack
of electron acceptors, which hampers the potential use of glycerol as
carbon source in bioprocesses (Booth et al., 2005; Shams Yazdani and
Gonzalez, 2008). However, recombinant strains producing fuels and
other high-value reduced chemicals using glycerol as substrate without
requiring external electron acceptors have been reported by inducing
the dormant, native 1,2-propanediol fermentative pathway in E. coli
(Dharmadi et al., 2006; Gonzalez et al., 2008; Murarka et al., 2008).
Dharmadi et al. (2006) demonstrated that glycerol can be converted
into reduced compounds such as ethanol and succinate by E. coli
under anaerobic and acidic conditions, and the activity of the formate
hydrogen-lyase and F(0)F(1)-ATPase systems were also found to facilitate the fermentative metabolism of glycerol for this process (Gonzalez
et al., 2008). Based on this investigation, they engineered E. coli for the
efcient conversion of crude glycerol to ethanol, (Shams Yazdani and
Gonzalez, 2008). Recombinant E. coli overexpressing glycerol dehydrogenase and dihydroxyacetone kinase and deleting fumarate reductase
and phosphate acetyltransferase produced ethanol-hydrogen from
unrened glycerol at yields exceeding 95% of the theoretical maximum
and specic rates in the order of 1530 mmol/g cell/h. These results are
superior to previous reported for the conversion of glycerol to ethanolH2 or ethanol-formate by other organisms and equivalent to those
achieved in the production of ethanol from sugar using engineered
E. coli strains (Shams Yazdani and Gonzalez, 2008). Another example
involved in converting glycerol to ethanol was reported with a novel
approach by employing oxygen as the electron acceptor in well dened
microaerobic culture conditions (Trinh and Srienc, 2009). In this study,
rational design with minimized metabolic functionality tailored to
efciently convert glycerol to ethanol using elementary node analysis
and adaptive laboratory evolution were implemented sequentially. As
a result, the strain converted 40 g/L glycerol to ethanol in 48 h with
90% of the theoretical ethanol yield (Trinh and Srienc, 2009).
2.3. Advanced biofuels production using recombinant E. coli as efcient
biocatalyst
Ethanol, as a traditional bulk chemical manufactured from various
feedstocks, is at present the main biofuel by volume as mentioned
above. However ethanol has a number of limitations as a fuel because
of its high hygroscopicity, low energy density, corrosiveness and
incompatibility with existing fuel infrastructure (Atsumi and Liao,
2008b; Zhang et al., 2011). Fortunately, other advanced biofuels, typically
higher alcohols (C4 and C5) such as butanol and isopentanol, terpenes
and fatty acid ethyl esters, have similar properties to current petroleum
based transportation fuels which could overcome the problems associated with the utilization of bioethanol (Atsumi and Liao, 2008b). Nevertheless, advanced fuels production by biological processes is still not
commercially feasible, except 1-butanol produced by Clostridium species (Jones and Woods, 1986), because of the absence of robust and
efcient biocatalysts. Therefore, much more research attention has
recently been dedicated to the development of microbial advanced
fuels. For example the fermentative and 2-keto acid pathways of
E. coli were employed to develop a catalyst for advanced fuels production
(Fig. 1) with 2-keto acid route being paid more attention for mediumchain alcohols production (Atsumi and Liao, 2008b; Zhang et al., 2011).
Signicant progress in microbial advanced fuels production through
metabolic engineering of E. coli is described below.
2.3.1. 1-Butanol and 1-propanol production
E. coli lacks some of the relevant pathways for advanced fuels
production and metabolic engineering has afforded the opportunity
to produce non-traditional biofuels through the construction of nonnative biosynthesis pathways (Atsumi and Liao, 2008b; Atsumi et al.,
2008b). 1-Butanol is attractive as an alternative biofuel with high
energy density and favorable compatibility with the potential to
1205
completely replace gasoline. Traditionally, 1-butanol was produced by

C. acetobutylicum together with acetone, butyrate and ethanol (Jones
and Woods, 1986). However, the complex physiology and absence of
genetic tools has limited the ability to improve the bacterium to a level
that application in a modern industrial process is feasible (Atsumi
et al., 2008a). Due to accumulated knowledge of the 1-butanol metabolic
pathway, it has become feasible to reconstruction of butanol pathway in
E. coli. In an example, Atsumi et al. (2008a) constructed a fermentatively
producing 1-butanol E. coli strain through transferring a set of six genes
(thl, hbd, crt, bcd, etfAB, adhE2) from C. acetobutylicum 1-butanol pathway
and deletion of the competing pathway. This engineered strain produced
1-butanol up to 552 mg/L from a rich medium under semi-anaerobic
conditions. While the product titer was low, the achievement has
paved a novel way for butanol production in a non-native host. To
achieve a higher titer and productivity, the substitution of two key
genes involved in 1-butanol pathway has been explored (Shen et al.,
2011). The replacement of the putative NADH-independent butyrylCoA dehydrogenase with the irreversible NADH-dependent transenoyl-CoA reductase, and Clostridium acetoacetyl-CoA thiolase with the
E. coli acetyl-CoA acetyltransferase, and deletion of the genes involved
in mixed-acid fermentation reactions, resulted in a strain that produced
30 g/L butanol with 70 to 88% of the theoretical yield when grown
aerobically followed by anaerobic fermentation. Similar approaches of
gene replacement and balancing of redox cofactors were employed by
Bond-Watts et al. (2011) to redirect more ux towards butanol production. This strain produced 4.65 g/L butanol with a yield of 28% from
glucose.
Recently alternative systematic approaches were implemented to
produce 1-butanol and 1-propanol by utilizing the amino-acid biosynthetic pathways have been reported (Fig. 1; Atsumi and Liao, 2008a;
Shen and Liao, 2008). 1-Propanol, a potential gasoline substitute, is a
general solvent with many industrial applications and can also be
converted to diesel fuels and propylene. However, it is difcult to
produce 1-propanol in signicant quantities using native organisms
(Shen and Liao, 2008). 2-Ketobutyrate, a precursor of isoleucine, not
only acts as a precursor of 1-propanol, but also can be converted to
1-butanol and 2-methyl-1-butanol via multi-steps enzymes reactions.
Therefore, E. coli was engineered to co-produce 1-butanol and 1propanol through this alternative route via 2-ketobutyrate. By overexpression of kivd (L. lactis), adh2 (S. cereviasiae), and the E. coli ilvA,
leuABCD, thrAfbBC, followed by deletion of the competing genes including tdh, metA, ilvI, ilvB, and adhE, an engineered strain was created (Shen
and Liao, 2008). This strain produced 2 g/L butanol and propanol with a
titer of nearly1:1 ratio but with an undesirable concentration, of
fermentative by-products. In another example, a more direct route to
synthesize 2-ketobutyrate via citramalate pathway was engineered by
directed evolution of Methanococcus jannaschii citramalate synthase
(CimA) to produce 1-propanol and 1-butanol (Atsumi and Liao,
2008a). Error-prone PCR and DNA shufing were carried out to enhance
the CimA specic activity and the resulting engineered E. coli strain
harboring the best CimA variant produced more than 3.5 g/L propanol
and 524 mg/L butanol. However production of other side products to
a signicant level remains a problem.
2.3.2. 2-Methyl-1-butanol and 3-methyl-1-butanol production
2-Methyl-1-butanol and 3-methyl-1-butanol, ve-carbon alcohols,
possess similar properties of lower vapor pressure and high energy
density. Compared to ethanol, they are more compatible with existing
fuel infrastructure and more suitable to replace gasoline. S. cerevisiae
can naturally produce 2-methyl-1-butanol and 3-methyl-1-butanol in
minor amounts as by-products, and some metabolic approaches have
been attempted to increase 2-methyl-1-butanol production in yeast
(Abe and Horikoshi, 2005). However, production of these ve-carbon
alcohols by E. coli is thought to be a more desirable process. Thus,
E. coli was engineered to produce 3-methyl-1-butanol and 2-methyl1-butanol through manipulation of the valine and leucine biosynthesis
1206
pathways, and the isoleucine biosynthetic pathway, respectively (Fig. 1)

(Cann and Liao, 2008). Using the isoleucine biosynthesis pathway, a
recombinant E. coli overexpressing native thrABC of the isoleucine pathway and the non-native ilvGM, ilvA, kivd and adh2 with the competing
pathways deleted resulted in a strain that produced 1.25 g/L 2methyl-1-butanol from glucose in 24 h. With a similar strategy,
Connor and Liao (2008) constructed a 3-methyl-1-butanol producing
E. coli strain via the native amino acid biosynthetic pathways. Upon
the removal of feedback inhibition and competing pathways, this strain
produced 3-methyl-1-butanol with a titer of 1.28 g/L from glucose in
28 h. Furthermore, this recombinant strain was employed to enhanced
3-methyl-1-butanol production by random mutagenesis and selection
(Connor et al., 2010). The resulting strain produced 9.5 g/L 3-methyl1-butanol with a yield of 0.11 g/g glucose when a two-phase fermentation process was used.
microbial organic acids are end-products, or at least natural intermediates in major metabolic pathways. Because of their functional
groups, organic acids extremely useful as starting materials for the
chemical, food and feed industries (Sauer et al., 2008). However, a
low efciency of bio-conversion of sugar to the desired product
together with a low yield and productivity limits the current market
for some organic acids dominated by the petrochemical industry.
Once a competitive fermentation technology based on strain improvement for these acids is established, the market for microbial produced
acids should increase (Bozell and Petersen, 2010). In this section, recent
critical advances in strain development for organic acid production
by metabolic engineering approaches using E. coli as a platform
are discussed with emphasis on lactic acid, succinic acid and 3hydroxypropionic acid as examples.
3.1. Lactic acid production
2.3.3. Isopropanol and isobutanol production

Isopropanol, one of the simplest secondary alcohols extensively
used in various applications, can be naturally produced by organisms
such as Clostridium. Although some attempts have been made to
improve the production ability of this strain, low titer and product
inhibition remain obstacles. Therefore, metabolic engineering of E. coli
for isopropanol production become a potential alternative industrial
route. Recently, Hanai et al. (2007) reported recombinant E. coli production of isopropanol using non-native fermentative pathways (Fig. 1).
Upon introduction of a set of genes in optimal combination from different sources, the pathway from acetyl-CoA via acetone to isopropanol
was established in an E. coli strain that produced 4.9 g/L isopropanol.
Moreover, this research group signicantly increased the titer up to
143 g/L with a molar yield of 67% (isopropanol/glucose) by the implementation of a gas trapping process, which promptly removed
isopropanol from culture broths to alleviate product toxicity to cell
(Inokuma et al., 2010). Notwithstanding the long fermentation time of
240 h, the recombinant E. coli produced the highest titer to date and is
much higher than the native clostridial strains. Introducing a similar
biosynthetic pathway described above, Jojima et al. (2008) constructed
a genetically engineered strain of E. coli that produced a 13.6 g/L
isopropanol from glucose under vigorous aerobic culture conditions.
Isobutanol has similar physical properties to isopropanol, but
possesses a higher octane number. As an intermediate in valine
biosynthesis, E. coli production of isobutanol was also engineered
through the synthetic nonfermentative pathways via 2-ketoisovalerate
as precursor (Fig. 1) (Atsumi et al., 2008b; Savrasova et al., 2011). To
increase 2-ketoisovalerate pool, the Bacillus subtilis alsS gene encoding
acetolactate synthase, and E. coli ilvCD gene encoding acetohydroxy
acid isomeroreductase, and dihydroxy-acid dehydratase, were overexpressed, and those pathways competing for pyruvate and cofactors
were deleted. Combined with overexpression of L. lactis kivd encoding
2-ketoacid decarboxylase, and S. cerevisiae adh2 encoding alcohol dehydrogenase, this engineered strain produced up to 22 g/L isobutanol at
86% of the theoretical yield after 110 h under micro-aerobic conditions
(Atsumi et al., 2008b). Comparative studies on the alcohol dehydrogenases (S. cerevisiae adh2, L. lactis adhA and E. coli yqhD) to convert
isobutyraldehyde to isobutanol in E. coli, both of which indicated that
overexpression of yqhD or adhA showed better results than adh2 in
isobutanol production (Atsumi et al., 2010). With a different culture process, Bastian et al. (2011) created an E. coli strain that produced 13.4 g/L
isobutanol with 100% theoretical yield under anaerobic conditions. This
strain was manipulated by overexpression of recombinant ketol-acid
reductoisomerase and alcohol dehydrogenase, with only NADH cofactor
specicity to achieve a redox balance under anaerobic conditions.
3. Organic acids
Organic aids are an important component of the building-block
chemicals that can be produced by microbial processes. Most
Lactate (D- or L-), an important organic acid, is a bulk chemical

increasingly used in the food and pharmaceutical industries in products
such as food, beverages and biodegradable polymers and currently
produced at 150,000 tons per year (Sauer et al., 2008). Previously,
Lactobacilli were commonly employed for industrial production of lactic
acid but had drawbacks of requiring complex growth nutrients, an
optically impure product, a mixed acid fermentation, and poor ability
to utilize pentoses (Wendisch et al., 2006). In order to expand the use
of lactic acid especially in biopolymers, it is necessary to improve the
optical purity of the acid required for polylactide use in bio-based plastics (Narayanan et al., 2004; Wee et al., 2006), and to reduce production
costs. Therefore, E. coli strains have been developed for effective production of D- and L-lactic acid using a metabolic engineering approach.
In E. coli, D-lactate is produced by homofermentative or
heterofermentative pathways together with NADH consumption in
order to achieve a redox balance under anaerobic culture conditions
(Fig. 2). However, in E. coli the pathway for production of L-lactate is
absent. E. coli has been successfully manipulated to produce optically
pure lactate with the potential for large scale commercial production
(Table 1; see reviews by Okano et al., 2010; Sauer et al., 2008;
Wendisch et al., 2006; Yu et al., 2011). In a rst successful example of
D- or L-lactate production by engineered E. coli, Chang et al. (1999)
created two strains, one which produced D-lactate with a titer of
62.2 g/L and one which produce L-lactate production with a concentration of 45 g/L. Two genes pta and ldhA were deleted and L-lactate dehydrogenase gene from Lactobacillus casei was overexpressed in the
L-lactate producing strain described above. Dien et al. (2002) engineered
E. coli strains to produce L-lactate with utilizing the mixtures of glucose
and xylose as substrates. A ptsG-negative but glucose-positive E. coli
strain was employed to produce L-lactic acid by deletion of the p and
d-ldh, with heterologous expression a L-lactate dehydrogenase gene.
The resulting recombinant strains produced L-lactate with a yield of
0.77 g/g sugar mixtures and low amounts of by-products. Similarly, a
series of D-lactate over-producing E. coli strains were created by many
investigators. Zhou et al. (2003) constructed derivatives of E. coli
W3110 for D-lactate production by elimination of competing pathways
including mutation genes of frdABCD, adhE, pB, and ackA. The resulting
strains produced D-lactate with the theoretical maximum yield of
2 mol/mol glucose. Moreover, the chemical purity and the optical purity
of above acid was up to 98% and 99%, respectively. However, these
strains showed a defect of incomplete metabolism of high sugar concentrations (more than 50 g/L). Accordingly, they constructed SZ132 strain
from an ethanologenic E. coli KO11, which rapidly fermented 100 g/L
sugars to produce D-lactate over 90 g/L in rich medium containing betaine as a protective osmolyte (Zhou et al., 2005). Because by-products
were increasingly produced and the poor performance in mineral salts
medium by the SZ132 strain, the strategies of metabolic evolution in
conjunction with genetic manipulations were carried out to yield an
over-producing strain E. coli SZ194 (Zhou et al., 2006). This strain
glucose
ptsG
1207
glycerol
galP
PEP
glpF
gldA
glk
glpK
PEP
pyruvate
glpABC
glpD
dhaKLM
glucose 6-P
pyruvate
DHAP
pck
2PEP
ppc
pps
lactate
pykA
pykF
ldhA
pyruvate
pyc
poxB
pflB
pdh
pta-ackA
maeA maeB
formate
acetyl CoA
acetate
adhE
ethanol
glyoxyate
C
mAB
2-oxo
gluta
rate
suc
cin
ate
B
hA
oA
sd
-C
yl
in
cc
su
fr
dA
te
ara
fum
aceA
e
BC
fu
citr
ate
ate
cet
loa
dh
oxa
m
aceB
malate
malat
rate
isocit
Fig. 2. Lactic acid and succinic acid pathways from glucose and glycerol in E. coli and enzymes involved in metabolic regulation (Blankschien et al., 2010; Wendisch et al., 2006;
Zhang et al., 2010; Zhou et al., 2011b). The three pathways for succinic acid production are indicated by the thick blue, red, and purple arrows, respectively. Relevant biochemical
reactions are represented by the names of the gene(s) coding for the enzymes (all E. coli genes unless otherwise specied): aceA: isocitrate lyase; aceB: malate synthase; ackA,
acetate kinase; adhE, acetaldehyde/alcohol dehydrogenase; dhaKLM, dihydroxyacetonekinase; frdABCD, fumarate reductase; fumABC, three isoenzymes of fumarases; gldA, glycerol
dehydrogenase; glk, glucose kinase; glpABC, anaerobic glycerol-3-phosphate dehydrogenase; glpF, glycerol facilitator; glpK, glycerol kinase; ldhA, D-lactate dehydrogenase; maeA/
maeB, NADH/NADPH malice enzymes, respectively; mdh, malate dehydrogenase; pck, phosphoenolpyruvate carboxykinase (E. coli or A. succinogenes); pdh, pyruvate dehydrogenase
complex; pB, pyruvate formate-lyase; poxB, pyruvate oxidase; ppc, phosphoenolpyruvate carboxylase; pps, PEP synthase; pta, phosphoacetyl transferase; ptsG, phosphotransferase
system; poxB, pyruvate oxidase; pyc, L. lactis pyruvatecarboxylase; pykA/pykF, pyruvate kinase.
produced D-lactate with a titer of 110 g/L, a yield of 0.95 g/g glucose
but only trace amounts of co-products in mineral salts medium
supplemented with 1 mM betaine. However, the chiral impurity of 5%
implicated that purication will be required adding to production cost.
A higher titer of D-lactate with 138 g/L was achieved by metabolic
engineering strain E. coli ALS974 (aceEF, p, poxB, pps, frdABCD) using a
dened medium and dual-phases process (aerobic growth and anaerobic
production) (Zhu et al., 2007). Overall productivity of 3.5 g/L/h, and an
overall yield of 0.86 g/g glucose were reported in a feed-batch fermentation. Noteworthy was that this strain necessitated the use of acetate to
form pyruvate during aerobic phase and that the reduction in the formation of succinic acid depended on the precise control of the acetate
concentration.
Zhou et al. (2012a) manipulated a wild-type E. coli B0013 by

deletion of eight genes (ackA-pta, pps, pB, dld, poxB, adhE, and frdA) to
over-produce D-lactate. Coupling lactate fermentation with cell growth
was employed and the cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/L/h and a yield of 86 g lactic acid/100 g
glucose which were 66% higher and at the same level compared to
that of the aerobic and oxygen-limited two-phase fermentation, respectively. These results have revealed an approach for improving production of fermentative products in E. coli. To the knowledge of the
authors, this was among the highest D-lactate levels reported for
engineered E. coli and shows favorable comparison with production by
lactate bacteria. Noteworthy is that strain B0013 was selected for its
properties of rapid growth, high tolerance to lactate, and ease of genetic
1208
manipulation compared to other standard strains such as E. coli K12 and

E. coli 3110. More recently, a genetic switching approach was developed
for highly efcient D-lactate production via ne-tuning of the D-lactate
dehydrogenase gene in which the chromosomal native promoter was
replaced with temperature-sensitive pR and pL promoters (Fig. 3)
(Zhou et al., 2012b). By combining the elimination of competing pathways for by-product and cofactor formation and temperature-shifting
the fed-batch fermentation process, a pure D-lactate titer of 122.8 g/L
from glucose was achieved with overall volumetric productivity of
4.32 g/L/h and the oxygen-limited productivity of 6.73 g/L/h. These
values are amongst the highest levels produced by a recombinant
E. coli strain without adding special substrates and obligatory anaerobic
conditions. (Zhou et al., 2012b). Recently, the fermentation performance
of this strain has been evaluated for D-lactate production in a 1000 Lfermentor and satisfactory results similar to those at laboratory scale
were obtained (unpublished data).
As mentioned above, crude glycerol has become an ideal feedstock
for the production of bio-based chemicals. Therefore, engineering
E. coli strains to produce lactate using glycerol as carbon source has
received attention. Mazumdar et al. (2010) created a recombinant
E. coli strain by overexpressing pathways involved in the conversion
of glycerol to D-lactate and the elimination those leading to the synthesis of competing by-products. Thus, an engineered homofermentative
E. coli strain was developed that converted 40 g/L glycerol to 32 g/L

with a yield of 85% of the theoretical maximum. Similarly a
metabolically engineered E. coli strain was created to produce D-lactate
from glycerol with a minimum of by-products in our lab (unpublished
data). With the elimination of by-product (formate, acetate, ethanol
and succinate etc) pathways and overexpression of lactate dehydrogenase gene, the resulting strain produced 114.4 g/L D-lactate with an average productivity of 3.95 g/L/h and yield of 0.78 g/g glycerol in 7-l
bioreactor with a modied two-phase fermentation process. Moreover,
only 1.8 g/L acetate was accumulated and no detectable succinate,
pyruvate, formate and ethanol were found (unpublished data). In addition, to expand the substrate range, Eiteman et al. (2008, 2009)
constructed two substrate-selective E. coli strains, one of which is
unable to consume glucose and one which is unable to consume xylose.
These two recombinant strains were used to simultaneously convert
xylose and glucose sugar mixtures to lactate by a co-fermentation strategy in a single process and the two-sugar mixture was efciently
converted into 37 g/L lactate with a lactate-sugar yield of 0.88 g/g.
Other products such succinic acid and acetate generated with lower
concentration during anaerobic conditions (Eiteman et al., 2009).
Although strain improvement of E. coli as a extensive platform was
achieved through genetic manipulation, a number of hurdles still need
to be crossed before the commercial production of D-lactate by an
D-lactate
Aerobic, 33
Oxygen-limited, 42
Repression
Active cIts
Byproducts
PR
Transcription
PL
ldhA
Glucose
Biomass
Pyruvate
Lactic acid
TCA
Inactive cIts PR
Byproducts
PL
ldhA
Glucose
Biomass
Pyruvate
Lactic acid
TCA
Fig. 3. An efcient strategy of D-lactate production through metabolic control approach in engineered E. coli with 30 g/L glucose in the initial M9 medium (Zhou et al., 2012a,
2012b). Lactate biosynthesis was inhibited in cells with competing pathways deleted when grown at 33 C under aerobic conditions due to inactive PR & PL promoters. When
cells were shifted to oxygen-limited and 42 C conditions, the promoters are activated and D-lactate production increases signicantly. Less substrate was used in TCA cycle and
by-product formation, and much more of the carbon ux was directed to the D-lactate pathway. In the top part, the dotted arrow shows the time when the culture was shifted
from 33 C to 42 C. The blue line indicates the time when the culture was shifted from the aerobic cultivation to the oxygen-limited production phase. Additions of glucose of
136.2 (A), 136.2 (B), 150.9 (C) and 136.2 g (D) were made to the bioreactor as indicated by arrows. In the bottom part, the red and grey arrows indicate the active pathways
and inactive pathways, respectively.
engineered E. coli strain can be applied. For example, the operating cost
including of separation and purication processes is one of bottlenecks
for the biotechnological production of optically pure lactic acid.
Weusthuis et al. (2011) suggested a solution where lactate is
co-produced with ethanol in an existing ethanol factory and separated
during distillation with minimal extra costs. The poor tolerance of lactic
acid producing E. coli strains to low pH requires application of large
amounts of Ca(OH)2, CaCO3 or other bases to maintain a neutral pH
during fermentation process and in some cases (special for calcium
ions) this could has become a big waste disposal problem in commercial
organic acids distillation. Development of acid tolerant E. coli strains by
directed evolution or systematic metabolic engineering would be preferable for commercial production.
3.2. Succinic acid production
Succinic acid was identied as one of the top 12 building block
chemicals by the U.S. Department of Energy (Bozell and Petersen,
2010). It is not only used in many elds such as agriculture, chemical,
food, and pharmaceutical industries, but also can offer a huge potential
alternative as a C4-dicarboxylic acid platform to be converted to other
chemicals such as 1,4-butanediol, tetrahydrofuran, -butyrolactone,
N-methyl pyrrolidinone, and 2-pyrrolidinone (Delhomme et al., 2008;
Sauer et al., 2008; Wendisch et al., 2006). The market potential for
succinic acid and its immediate derivatives was estimated to be up to
245,000 tons per year (Bozell and Petersen, 2010) but most commercially available succinic acid is presently produced by a petrochemical
process which generated negatively economical and ecological impacts.
Excitingly, the signicant progress in the development of an economically competitive route of succinic acid production from renewable resources has been made. Commercial succinic acid production via the
industrial biotechnology route has been reported by the Requette
(http://www.dsm.com/en_US/downloads/media/12e_09_dsm_and_
roquette_commercialize_bio_based_succinic_acid.pdf.) and Bioamber
(http://www.bio-amber.com/press_releases.php.) companies. Many
literature reports on succinic acid production from renewable substrates have been published including a number of processes based on
engineered E. coli strains (Jantama et al., 2008a; Lee et al., 2005).
E. coli naturally produces succinic acid in a minor amount as an
intermediate of the central metabolism or as a fermentation endproduct via the reductive tricarboxylic acid (TCA) branch (Fig. 2). There
are three alternative routes to form succinic acid: the PEP-pyruvateoxaloacetate node (the reductive TCA branch), the oxidative TCA branch
and the glyoxylate shunt, respectively (Fig. 2), each of which is involved
in redox balance and energy regeneration. The formation of 1 mol of
succinic acid via the reductive pathway is calculated to consume 2 mol
of NADH and 1 mole of CO2, whereas 1 mol of glucose can furnish only
2 mol of NADH through the glycolytic pathway (Fig. 2). Therefore, the
redox balance and high succinic acid yield are closely tied to the pool of
NADH and these existing complex metabolic networks and regulation
mechanisms requires alternative strategies of metabolic engineering
of E. coli for succinic acid production to be considered.
A feasible alternative strategy is to engineer E. coli to anaerobically
overproduce succinic acid by amplication of phosphoenolpyruvate
carboxylase (PPC) which catalyzes PEP to oxaloacetate (OAA) with
CO2 consumption (Kim et al., 2004; Millard et al., 1996). Stols and
Donnelly (1997) investigated whether the amplication of the malic
enzyme gene would restore fermentative metabolism of glucose and
produced succinic acid as a major fermentation product in E. coli
NZN111, a strain unable to ferment glucose because of inactivation of
the genes ldhA and p. When recombinant E. coli NZN111 harboring
malic enzyme gene was cultured in LB medium containing 20 g/L sorbitol as a more reduced carbon source under a CO2 atmosphere, 10 g/L of
succinic acid was produced (Hong and Lee, 2002). These results were
consistent with the prediction of in silico metabolic ux analysis (Lee
et al., 2002), which indicated that redox balancing was important for
1209
the enhanced production of succinic acid. Subsequently, inactivation

of ptsG gene, encoding the membrane-bound protein involved in glucose transport, not only partly restored the ability of NZN111 strain to
grow fermentatively on glucose, but also allowed this mutant strain to
yield higher succinic acid concentrations under anaerobic conditions
(Chatterjee et al., 2001). Using an optimized dual-phase fermentation,
the recombinant strain NZN111 (ptsG inactived, pyc gene overexpressed) produced 99.2 g/L succinic acid with an overall yield of
110% on glucose and productivity of 1.3 g/L/h (Vemuri et al., 2002),
which is the highest titer produced by a recombinant E. coli reported
in the literature. Furthermore, overproduction of succinic acid was
investigated by inhibition of byproduct formation (Lee et al., 2005),
diversion of NADH and provision of additional precursor for succinic
acid synthesis (Sanchez et al., 2005), as well as optimization of the
fermentation process (Jiang et al., 2010; Wu et al., 2007). Interestingly,
various studies indicate that CO2, which is incorporated into the carbon
backbone to form OAA from PEP via carboxylation of PPC, affected
succinic acid accumulation through the reductive TCA branch under
anaerobic fermentation process (Lu et al., 2009).
Systems metabolic engineering was applied to improve succinic acid
production. Using the combination of genome-scale in silico optimization and metabolic ux analysis, remarkable improvement of succinic
acid production in E. coli was achieved with a yield of 1.29 mol/mol
glucose (Wang et al., 2006). In another example, Lee et al. (2005)
compared the genomes of E. coli and succinic acid-overproducing
Mannheimia succiniciproducens, and constraints-based ux analysis
was carried out to improve the succinic acid production of E. coli,
which strategy increased the succinic acid production by more than
sevenfold and the ratio of succinic acid to fermentation products by
ninefold. Although succinic acid yield was not signicantly increased,
the results suggest that this approach can be an efcient way of developing strategies for strain improvement.,
Recently, directed evolution in combination with genetic engineering was carried out to achieve a succinic acid overproducing
strain with energy-conserving pathways, in which pck was cloned and
evolved to the major carboxylation pathway (PEP + CO2 + ADP =
oxalaacetate + ATP) for succinate production with the CO2 xation
and generation of ATP. In addition the native PEP-dependent
phosphotransferase system (PTS) for glucose uptake was inactivated
(Jantama et al., 2008a; Zhang et al., 2009). Owing to no additional reducing equivalent consumed from glucose to succinic acid, this evolved
strain not only increased the pool of PEP available for redox balancing
but also increased energy efciency, thus leading to enhanced succinic
acid production with a ATP-generating pathway, which naturally exists
in succinic acid-producing rumen bacteria (Lee et al., 2006). Moreover,
this evolved strain was further engineered to improve succinic acid
production by deletion of the genes encoding the acetate, malate, and
pyruvate pathways. The strain produced 700 mM succinic acid with a
high yield of 1.5 mol/mol glucose (Jantama et al., 2008b), which are
comparable to the best natural succinic acid-producing rumen bacteria
such as Actinobacillus succinogenes. In another similar example, to maintain the redox and ATP balance, Singh et al. (2011) cloned the gene
encoding the ATP-generating PEPCK enzyme in combination with elimination of ethanol and acetate forming to improve succinic acid synthesis
in the ldhA, pB, ptsG mutant strain, and the resulting E. coli strains
produced succinic acid with a 60% increase compared to the a control
strain.
Another strategy of metabolic engineering has been carried out to
overproduce succinic acid under aerobic conditions (Lin et al., 2005a,
2005b, 2005c). In a successful example, they constructed two aerobic
central metabolism routes for succinic acid production, one of which
is a glyoxylate shunt and the other is an oxidative TCA branch for
aerobic succinic acid production, by inactivation of genes sdhAB, iclR,
poxB, and ackA-pta (Lin et al., 2005c). The resultant strain was further
engineered by deletion of ptsG and overexpression of pepc gene from
Sorghum vulgare, to give an over-producing strain. Using a fed-batch
1210
process, the above strain produced 58.3 g/L succinic acid with a yield
of 0.94 mol/mol glucose and a productivity of 1.08 g/L/h under strictly aerobic conditions (Lin et al., 2005a), which pointed to a promising
system for large-scale succinic acid production. Alternatively, an
engineered E. coli strain, using sucrose as carbon source to aerobically
produce succinic acid with a mol yield up to of 1.90, was achieved by
overexpression of scr genes (scrK, Y, A, B, and R genes converting sucrose to -D-fructose and -D-glucose 6-phosphate) and Lactococcus
lactis pyc gene (Wang et al., 2011b).
To expand the range of available substrates and efciently convert
inexpensive feedstock to high value bio-based products such as
succinic acid, glycerol, sucrose, fructose and/or a mixture sugar were
tested to produce succinic acid by various genetically modied E. coli
strains (Blankschien et al., 2010; Kang et al., 2011; Wang et al., 2011a,
2011b). In a successful example, Blankschien et al. (2010) created an
E. coli strain by overexpression of the L. lactis pyruvate carboxylase
gene and by blocking pathways for the synthesis of by-products,
which produced succinic acid with a yield of about 0.69 g/g glycerol
from 20 g/L glycerol in 72 h, on par with the use of glucose as a feedstock. Similarly, Zhang et al. (2010) created an E. coli strain able to
efciently convert glycerol to succinic acid without introduction of
non-native E. coli genes. Upon mutational activation of phosphoenolpyruvate carboxykinase gene (pck*), and inactivation of ptsI (encoding
PtsI of the phosphorelay system) and pB gene (encoding pyruvate
formate-lyase), the resulting engineered strain converted 128 mM
glycerol to 102 mM succinic acid with 80% of the maximum theoretical
yield during anaerobic fermentation in mineral salts medium. Nevertheless, low growth, poor fermentation performance and long culture
period are major limitations for these strains. In another example,
21.07 g/L succinic acid and 0.54 g/L polyhydroxyalkanoate (PHA) was
produced simultaneously from a mixture of glycerol and fatty acid by
a modied E. coli strain created by Kang et al. (2011). However, similar
to the problem for lactate production mentioned above, the need for
medium neutralization process and the poor acid tolerance of the cell
are major disadvantages for succinic acid production by metabolically
engineered E. coli strains.
3.3. Production of other organic acids
In addition to lactic acid and succinic acid as discussed above,
other organic acids such as pyruvate, acetate and malate are also
important bulk chemicals and have a wide range of industrial applications. Currently, production of these acids is mainly by traditional
petrochemical-based routes and/or biotechnological processes by wild
type organisms. However, some investigations have recently reported
that metabolically engineered E. coli strains have been employed to
overproduce these acids In one example, Causey et al. (2003) created
an excellent E. coli strain by sequential genetic modication, which
can efciently convert sugar to acetate as the primary product. Upon
introduction of the ldhA, pB, frdBC, atpFH, adhE and sucA genes mutations together with the blocking of native fermentation pathways,
oxidative phosphorylation and TCA function, the resulting strain
named E. coli TC36 produced up to 878 mM acetate with 75% of the
maximum theoretical yield from a mineral salts medium containing
glucose as carbon source. Moreover, the same group further genetically
manipulated the TC36 strain by deletion of poxB and ackA genes to yield
pyruvate strain E. coli TC44 that accumulated up to 749 mM pyruvate
with a yield of 0.75 g/g glucose (Causey et al., 2004). All major nonessential pathways consuming pyruvate in this multiple mutated strain
were absent and the utilization of pyruvate for cell growth was reduced.
In an another example, Tomar et al. (2003) constructed an overproducing pyruvate E. coli strain by deletion of the gene encoding the
E2p subunit of the PDHC and ADH encoding gene in a PEP carboxylase
decient strain. Using this genetically engineered strain, 425 mM
pyruvate was obtained with a yield of about 0.60 to 0.74 g/g glucose
but some byproducts such as acetate and lactate were also detected.
Therefore, metabolic engineering was further carried out to direct

more carbon to pyruvate by elimination of intracellular conversion
of pyruvate to acetyl-CoA, PEP, acetate and lactate. Hence an
overproducing E. coli was achieved by which 1 mol glucose was
converted to 1.78 mol pyruvate, with titers as high as 110 g/L (Zelic
et al., 2004). Zhu et al. (2008) created an overproducing pyruvate
E. coli strain ALS1059 by mutations in the aceEF, p, poxB, pps, ldhA,
atpFH and arcA genes which encode, respectively, the pyruvate
dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase,
phosphoenolpyruvate synthase, and lactate dehydrogenase. Using this
strain, 90 g/L (more than 1 M) of pyruvate production with an overall
productivity of 2.1 g/L/h and yield of 0.68 g/g was achieved with the
fed-batch process.
Furthermore, E. coli also has been engineered to produce some
high-value organic acids such as shikinic acid (Escalante et al., 2010;
Johansson et al., 2005), glucaric acid (Moon et al., 2009, 2010) and 3hydroxypropanoic acid (3-HP) (Mohan Raj et al., 2009; Rathnasingh
et al., 2009), which generally are secondary metabolites that are produced in much lower amounts than the previously mentioned acids.
These organic acids are important compounds used in many elds
such the pharmaceutical and chemical industries.
4. Amino acids production
Amino acids are important bioproducts with extensive industrial
applications in the antibiotic, pharmaceutical, animal feed, and cosmetic
industries as well as in food additives. More than two million tons of
amino acids are produced annually (Park and Lee, 2008) with annual
growth rate of 57% (Lee et al., 2007) including more than 500 tons of
L-valine, 8000 tons of L-phenylalanine, 1,000,000 tons of L-glutamate
and, and over 4000 tons of L-threonine by fermentation (Ikeda, 2003).
Traditional strategies of strain development for amino acid production
were multi-round random mutation and selection procedures. Because
of the complicated and highly regulated metabolic network, these
approaches have played the major role in development of industrial
amino acid producers in recent decades. However, such approaches
inevitably or unintentionally result in unwanted mutations not directly
related to the target metabolite which have undesirable effects on the
physiology of the organism and retard growth (Park et al., 2007).
Targeted metabolic engineering of E. coli and other organisms has
been applied to strain improvement with the purpose of modifying
specic genes and pathways to enhance production of a desired product
(Clomburg and Gonzalez, 2011; Lee et al., 2004; Nikel et al., 2010; Wang
et al., 2011a; Yi et al., 2002). These rational engineering strategies have
played a critical role in microbial improvement but the local pathways
or bioreactions of engineered cell are usually emphasized and
genome-wide, global metabolic network is often neglected. In recent
years, with the advances of omics technology and genome-scale computational biology, systems metabolic engineering (systems biology
combined with metabolic engineering), has been applied in strain
improvement. By integrating information of entire metabolic and regulatory networks (Kumar and Shimizu, 2011; Lee et al., 2007; Park and
Lee, 2008; Park et al., 2007, 2011; Schaub et al., 2008), the problems
associated with random mutation and the difculty of rationally engineering the complex and highly regulated metabolic network can be
overcome.
Currently, E. coli has been extensively used for the production of
various amino acids (Ikeda, 2003) (Table 1), such as L-valine (Park
et al., 2007, 2011), L-phenylalanine (Baez-Viveros et al., 2004, 2007;
Gerigk et al., 2002), L-alanine (Lee et al., 2004), and L-threonine (Dong
et al., 2011; Lee et al., 2003a, 2009; Song et al., 2000). The metabolic
pathway of important amino acids and their regulator mechanism in
E. coli, are shown in Fig. 4. Here, some recent representative examples
are highlighted with particular emphasis on the advances of rational
metabolic engineering and system-level engineering approaches to
improve amino acid producing E. coli strains.
1211
Pentose phosphate pahwtway

Glucose
Ribulose 5-phosphate
Glycolysis
Erythrose 4-phosphate
ppc
aroF
aroG
aroH
Phosphoenolpyruvate
Pyruvate
Oxaloacetate
3-deoxy-D-arabinoheptulosonete 7-phosphate
ilvBN
aspC
ilvGM
TCA
aroB
ilvIH
L-Aspartate
3-Dehydroquinic acid
2-Acetolactate
thrA
-Lysine
metL
lysC
Aspartyl phosphate
aroD
ilvC
3-Dehydroshikimic acid
2,3-Dihydroxyisovalerate
aroE
asd
ilvD
Shikimatic acid
Aspartate semialdehyde
thrA
metL
-Leusine
aroL
aroK
2-Ketoisovalerate
Shikimic acid-3-phosphate
ilvE
L-Homoserine
aroA
thrB
L-Valine
Homoserine phosphate
3-Enolpyruvyl-shikimate
-5-phosphate
Cystathionine
aroC
thrC
L
Leusine
responsive protein
Homocysteine
-Threonine
Chorismate
sm
trpE
Prephenate
p
L
tdcC
-Threonine
livJ
-Valine
-Tyrosine
-Phenylalanine
pheA
-Tryptophann
ygaZH
Transproter
Exporter
-Threonine
-Methionine
L
rhtA
rhtB
rhtC
Transproter
typA
Exporter
-Valine
Fig. 4. The biosynthetic pathway and regulatory circuits involved for L-valine, L-threonine, L-methionine, and aromatic amino acids in E. coli (Dong et al., 2011; Lee et al., 2007; Park
and Lee, 2008). The shaded boxes represent genes encoding enzymes catalyzing the corresponding reactions. The thick grey arrows indicate the glycolysis pathway and pentose
phosphate pathway. The thick red arrows indicate the related catalytic steps in the amino acid pathways. The blue dotted arrows indicate the feedback inhibition and the black
dotted arrows indicate the feedback activation.
4.1. L-Threonine production

L-Threonine is one of the three major amino acids produced by fermentation processes (Takors et al., 2007), belonging to the aspartate
family of amino acids. The biosynthesis pathway of L-threonine
consists of ve enzymatic reactions from L-aspartate in E. coli (Fig. 4),
in which the rst reaction step catalyzed by three aspartokinase isoenzymes are critical for L-threonine formation. Aspartokinase I encoded
by thrA is inhibited by Thr, and its synthesis is repressed by Thr and
L-isoleucine. Aspartokinase II is encoded by metL and its synthesis is
repressed by L-methionine. Aspartokinase III encoded by lysC is inhibited
by L-lysine and L-lysine also inhibits its synthesis. Furthermore,
aspartokinase I and II are bifunctional enzymes with two catalytic
domains, one for aspartate kinase activity and the other for homoserine
dehydrogenase activity. Therefore, they are also named as aspartokinase
Ihomoserine dehydrogenase I and aspartokinase IIhomoserine dehydrogenase II (Fig. 4). In addition, the homoserine kinase encoded by
thrB gene is subject to feedback inhibition by L-threonine as homoserine

dehydrogenase I.
In earlier studies, an attempt was made to produce L-threonine by
E. coli strains through strain improvement or optimizing the fermentation process (Furukawa et al., 1988; Shiio and Nakamori, 1969;
Song et al., 2000). More recently, strategies of systems metabolic engineering have been successfully implemented for L-threonine production and revealed a more powerful tool for the enhanced production
of a desired metabolite. Typically, combined genome, transcriptome,
and proteome analyses were employed to elucidate the biosynthetic
and regulatory mechanisms of physiological changes between a L-threonine overproducing E. coli strain and the wild-type strain (Lee et al.,
2003a). Combined omics analyses indicated that genes involved in the
glyoxylate shunt, the tricarboxylic acid cycle and amino acid biosynthesis were signicantly upregulated, whereas many ribosomal protein
genes were downregulated. Furthermore, two signicant mutations in
the thrA and ilvA genes were identied, which are essential for
1212
overproduction of L-threonine. In a similar example, an overproducing

L-threonine E. coli strain was developed with a systems metabolic
engineering approach (Lee et al., 2007). Initially, negative regulation
including feedback inhibition and transcriptional attenuation regulation was removed by targeted metabolic engineering. Then, the target
genes to be engineered were identied by transcriptome proling combined with in silico ux response analysis, and their desirable expression levels were manipulated accordingly. The nal engineered E. coli
strain was able to produce L-threonine with a signicant high yield of
0.393 g L-threonine/g glucose, and 82.4 g/L L-threonine by fed-batch
fermentation. Subsequently, Lee et al. (2009) reported reengineering
of a reduced-genome E. coli for L-threonine production. Using strain
MDS42 lacking 14.3% of its chromosome as host (Posfai et al., 2006), a
feedback-resistant L-threonine operon was over-expressed, the genes
tdh encoding L-threonine dehydrogenase, tdcC and sstT encoding
L-threonine transporters, respectively, were deleted, and a L-threonine
exporter encoded by the mutated gene rhtA23 was introduced. The
resulting strain MDS-205 showed an 83% increase in L-threonine production, compared to a wild-type E. coli strain MG1655 engineered
with the absolutely same modications. Moreover, transcriptional analysis revealed that elimination of nonessential genes can increase the
productivity of an industrial strain, by reducing the metabolic burden
and improving the metabolic efciency of cells. These examples also
indicated that a systems metabolic engineering approach can be
employed to obtain an industrially competitive strain.
4.2. L-Valine production
L-valine, a branch-chain amino acid, has been used in pharmaceuticals,
cosmetics, and feed-additive industries and produced by random mutant
or rational engineering strains such as Serratia and Corynebacterium
glutamicum (Elisakova et al., 2005; Plachy, 1975). However, no publications have reported L-valine production by E. coli until 2007, because
E. coli strain has a more complicated regulatory mechanism for L-valine
biosynthesis than any other microorganism (Park et al., 2007). In E. coli,
three isoenzymes acetohydroxy acid synthase (AHAS I, II, and III, encoded
by ilvBN, ilvGM, and ilvIH, respectively) catalyze pyruvate to 2acetolactate, the critical step for L-valine pathway (Fig. 4). Expression of
the ilvGMEDA operon is inhibited by all three amino acids L-valine,
L-leucine, and L-isoleucine mediated by transcriptional attenuation,
whereas the expression of the ilvBN operon is affected by attenuation
mediated by L-valine and L-leucine. Furthermore, the global regulator
leucine responsive protein (Lrp) activates the expression of ilvIH and
L-valine exporter genes, and negatively affects the expression of the
ilvGMEDA operon and L-valine transporter genes (Fig. 4). Park et al.
(2007) constructed a genetically engineered E. coli strain capable of
overproducing L-valine using available information, transcriptome analysis and in silico genome-scale simulation. With this systematically
metabolic engineered strain, an impressively high L-valine yield of
0.378 g/g glucose was achieved. Subsequently, this research group
engineered a high L-valine tolerant E. coli W strain to overproduce Lvaline (Park et al., 2011). After the authors removed negative regulatory
circuits and amplied a transporter and Lrp, a positive-acting global
regulator, they obtained an engineered strain, which was able to
produce 60.7 g/L L-valine without other byproduct formation in 29.5 h
by fed-batch culture. This value is higher than that previously reported
for industrial C. glutamicum or other E. coli strains (Blombach et al.,
2007, 2008).
4.3. L-Phenylalanine production

The aromatic amino acids such as L-tryptophan, L-tyrosine and
are used in diverse commercial applications. Among
them, L-phenylalanine has attracted much attention due to its demand
in the food industry for an the synthesis of the low-calorie sweetener
aspartame additive (Gerigk et al., 2002; Ikeda, 2003; Sprenger, 2007a,
L-phenylalanine,
2007b). E. coli synthesizes L-tryptophan, L-tyrosine and L-phenylalanine

via a common metabolic pathway, known as the shikimatic acid pathway. Phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P)
are condensed to form 3-deoxy-D-arabinoheptulosonate-7 phosphate
(DAHP), and transformed by multiple steps to form chorismate (Fig. 4).
Chorismate is converted to L-tyrosine and L-phenylalanine by chorismate
mutase-prephenate dehydrogenase and chorismate mutase-prephenate
dehydratase, respectively (Sprenger, 2007a). L-Tryptophan is synthesized through ve steps from chorismate (Fig. 4; Sprenger, 2007a,
2007b). The precursors for aromatic biosynthesis, PEP and erythrose4-phosphate (E4P) are formed via glycolysis and the pentose phosphate
pathway, respectively.
A strategy to alleviate inhibition by regulatory circuits, to increase
the availability of precursors and to overcome the rate-limiting steps
can yield a means to improve aromatic acid production. Some of these
strategies have been successfully applied to develop improved
aromatic acid producing strains (Flores et al., 1996; Sprenger, 2007b).
Baez-Viveros et al. (2004) created an overproducing L-phenylalanine
strain using metabolic engineering and protein-directed evolution
strategies. In their report, the simultaneous overexpression of
transketolase, feedback-resistant inhibition DAHP synthase, and
evolved chorismate mutase-prephenate dehydratase combined with
PTS inactivation in E. coli resulted in the enhanced L-phenylalanine
production with yield of 0.33 g/g and productivity of 40 mg/g-dcw/h
from glucose. Recently, Zhou et al. (2011a) reported that a recombinant
E. coli BR-42 strain harboring the L-phenylalanine synthesis-related
plasmids produced L-phenylalanine up to 57 g/L with a high productivity of 1.2 g/L/h using two-phase feeding strategy. Interestingly, the
recombinant E. coli strain was originally developed to produce Lphenylalanine using glycerol as an inexpensive and abundant sole carbon source with a yield of 0.588 g/g glycerol under aerobic conditions
(Khamduang et al., 2009).
Systems-level metabolic engineering has also been carried out to
improve aromatic amino acid production by E. coli (Baez-Viveros
et al., 2007; Kedar et al., 2007; Polen et al., 2005). Proteomic analysis
was carried out to investigate metabolic changes in the aromatic acid
biosynthesis by pyk-F mutant E. coli and the wild type strains, and the
results revealed that aromatic amino acid overproduction accompanied
signicant changes in the synthesis level of key metabolic enzymes
involved in aromatic acid biosynthesis and degradation pathway
(Kedar et al., 2007). Moreover, metabolic transcription analysis was
performed to detect transcriptional responses with an engineered
L-phenylalanine overproducing E. coli, in which the PTS transporter
gene was deleted and key genes encoding DAHP synthase, transketolase
and chorismate mutaseprephenate dehydratase were overexpressed
(Baez-Viveros et al., 2007). The results showed a set of genes encoding
carbohydrate transporters and gluconeogenic and fermentative enzymes
were up-regulated in the overproducing strain and indicated that reverse
engineering approaches are feasible for the further improvement of
L-phenylalanine production strains.
4.4. Production of other amino acids

L-Tryptophan and L-tyrosine, the other two aromatic amino acids,
also have been overproduced by E. coli strains using the similar metabolic engineering approaches (Chavez-Bejar et al., 2008; Olson et al.,
2007; Zhao et al., 2011). In additional, E. coli was engineered by genetic
manipulation to successfully produce some other amino acids and their
derived compounds/derivatives, such as L-alanine, L-methionine,
L-ornithine and anthranilate (Balderas-Hernandez et al., 2009; Kromer
et al., 2006; Lee and Cho, 2006; Lee et al., 2004; Wada et al., 2007).
One successful example of an engineered E. coli overproduced L-alanine
with a titer of 114 g/L and a yield of 0.95 g/g by implementation of
metabolic evolution strategy combined with metabolic engineering
(Zhang et al., 2007b).
These examples above show how various metabolic engineering

strategies can be employed to increase E. coli capability to produce
desired amino acids and some of the engineered strains have been
commercialized, which further indicates that E. coli strains can
become the basis of an industrial platform for amino acids production.
5. Sugar alcohols
Sugar alcohols, including xylitol, mannitol, and sorbitol, are generally
produced by a wide variety of microbes and plants as secondary metabolites. They have characteristics similar to sugar and demonstrate many
uses in different applications in the pharmaceutical, food, and chemical
industries (Akinterinwa et al., 2008). Traditionally, sugar alcohols are
produced mainly by a chemical process via catalytic hydrogenation on
activated nickel catalysts. However, costly downstream processing and
non-specic reaction conditions limit further development. Recently,
enzymatic synthesis routes and whole cells catalytic processes have
become more attractive owing to high selectivity and environmentally
friendly approaches. Here we will only concisely discuss the recent
efforts to metabolic engineering E. coli for xylitol and mannitol
production.
Xylitol, is a natural sweetener with a annual market value of about
$340 million that places it as a top value added chemical from biomass
(Kadam et al., 2008), but most of this is still produced by the chemical
transformation route. Genetically modied Candida tropicalis and
S. cerevisiae strains were reported to produce xylitol by process optimization (Ko et al., 2006; Toivari et al., 2007). Recent successful examples
demonstrated that genetically engineered E. coli can also produce
xylitol with a high titer from different carbon sources (Akinterinwa
and Cirino, 2011; Cirino et al., 2006; Sakakibara et al., 2009). Previously
Suzuki et al. (1999) obtained a recombinant E. coli strain by heterologous amplication of the C. tropicalis xyrA gene encoding a D-xylose
reductase. Using this strain, 13.3 g/L xylitol was achieved from a mixture of xylose (50 g/L) and glucose (5 g/L) after 20 h. Subsequently,
an engineered E. coli strain converting L-arabinose to xylitol was created
by introducing a pathway from L-arabinose to xylitol (Sakakibara et al.,
2009). Addition of glycerol as co-substrate improved the xylitol titer to
14.5 g/L with a yield of 0.95 g/g L-arabinose.
In other successful examples, Cirino and coworkers (Akinterinwa
and Cirino, 2011; Chin and Cirino, 2011; Chin et al., 2009; Cirino et al.,
2006; Khankal et al., 2008) created various recombinant strains able
to overproduce xylitol by the implementation of a range of metabolic
engineering approaches including by overexpressing the key enzymes
in xyltiol biosynthetic pathway, by increasing the availability of reduced
equivalents pool, and by mutating cyclic AMP receptor protein to cyclic
AMP-independent one (crp*). Upon introducing a NAD(P) + cofactor regenerative system by glucose oxidation coupled to xylose reduction via
the NADPH-dependent xylose reductase, xylitol was produced from xylose with the maximum theoretical yield of 4 moles of xylitol per mole
of glucose consumed under anaerobic and nonrespiratory conditions
(Akinterinwa and Cirino, 2011). In another fed-batch cultivation
study, a xylitol titer of 56 g/L was achieved in a strictly dened mineral
salts medium containing a mixture of xylose and glucose by an E. coli
strain overexpressing xylose transporters and a C. boidinii xylose reductase coupled to deletion of the xylB gene (Khankal et al., 2008).
The annual market of mannitol, a six-carbon sugar alcohol, is
estimated to be up to 40,000 tons (Kaup et al., 2004). Many organisms
including bacteria and plants can produce mannitol and Candida
magnoliae has been used for the industrial production (Lee et al.,
2003b). However, metabolic engineering efforts to enhance mannitol
production by E. coli offer an alternative route (Heuser et al., 2009;
Kaup et al., 2004). An E. coli strain able to produce mannitol from fructose was constructed by expressing a Leuconostoc pseudomesenteroides
mannitol dehydrogenase (MDH) and Mycobacterium vaccae formate
dehydrogenase (FDH) (Kaup et al., 2004). Similar to the xylitol production by E. coli discussed above, the combination of NADH generated by
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FDH from formate and the regeneration of NAD+ by MDH from fructose
in the recombinant cells resulted in a cofactor cycle system that
supported mannitol production (Kaup et al., 2004). In additional, a
glucose facilitator transporter was expressed to increase the efciency
of fructose uptake. The resulting strain was used to produce about
91 g/L mannitol with a molar yield of about 90% when fed-batch cultivation was conducted (Kaup et al., 2004). Furthermore, overexpression
of a putative permease from L. pseudomesenteroides resulted in an
increase of mannitol yield by 20%, whereas increasing available NAD
pool and controlling CO2 concentration had no signicant impact on
mannitol productivity (Heuser et al., 2009).
6. Biopolymer and monomers production
Currently, petroleum-based polymers are preferred for household
and industrial applications. However the growing awareness of
sustainability of supply and environmental issues, bio-based polymers
from renewable feedstocks, possessing biodegradable, biocompatible
and multipurpose features, are more attractive alternatives to traditional
materials. Recently, metabolically engineered E. coli has been employed
to produce various biopolymers and monomers (Lee et al., 2011a; Zeng
and Sabra, 2011). Many building block chemicals such as lactic acid,
succinic acid, fumaric acid, itaconic acid, and diols including 1,3propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol
(2,3-BDO) and 1,4-butanediol (1,4-BDO) can be used as a monomer
for polymers. In addition, biopolymers such as polyhydroxyalkanoates
(PHAs) and polylactic acid (PLA) are generally used for biodegradable
polymers and can be microbiologically produced. Here, we briey
describe E. coli as a robust biocatalyst for monomers production and
polymer synthesis, together with the limitations and opportunities.
Lactate and succinic acid production have been discussed in Sections 3.1
and 3.2 and will not be dealt further.
6.1. 1,3-Propanediol and 1,2-propanediol
1,3-PDO, one of most important and attractive monomers for the
synthesis of polyesters for fabric and textile applications, was traditionally produced from fossil resources. However, 1,3-PDO produced
from glucose by a recombinant E. coli strain has been commercial
for a number of recent years (Nakamura and Whited, 2003). Although
this biological route agrees with code of sustainability as well as the
high selectivity and the mild operation conditions, the high cost of
the process limits ability of compete with production via the chemical
route. Therefore, strain improvement by metabolic engineering has
attracted much attention to improve economical production of
1,3-PDO recently.
1,3-PDO pathway is a two-step sequential enzymatic process from
glycerol. Initially, glycerol is converted to an intermediate 3hydroxypropionaldehyde by glycerol dehydratase encoded by the
gene dhaB, then is reduced to 1,3-PDO by an NADH-dependent
1,3-propanediol oxidoreductase encoded by the gene dhaT. Because
of absence of a dha regulon, E. coli is unable to produce 1,3-PDO
(Tong et al., 1991). Owing to the ability of K. pneumoniae and Clostridium
species to anaerobically convert glycerol to 1,3-PDO and availability of
the cheap glycerol, many attempts have been made to increase
K. pneumoniae or C. pasteurianum 1,3-PDO yield and production using
various metabolic and fermentation strategies (Jun et al., 2010; Seo
et al., 2009; Xu et al., 2009). However, the most successful process
described to date by recombinant E. coli production of 1,3-PDO. DuPont
and Genencor International Inc. created an industrial E. coli strain
overproducing 1,3-PDO from glucose by introduction of glycerol pathway from S. cerevisiae and 1,3-PDO pathway from K. pneumonia
(Nakamura and Whited, 2003). The resulting strain produced 1,3-PDO
with a titer of more than 130 g/L, which is the highest titer reported to
date. However, little detailed scientic literature about this work has
been published and only patents were disclosed (Emptage et al., 2003;
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Nakamura et al., 2000). Given the importance and signicance of this

process, companies are hesitant to reveal full details of their process.
Using a similar approach, other research groups have constructed various E. coli strains for 1,3-PDO production from glucose (Jin and Lee,
2008), but the titer was lower than that reported in the patent literature.
Jin and Lee (2008) deleted six genes (galR, glpK, gldA, ldhA, lacI, tpiA) to
create an recombinant E. coli strain, which produced 43 g/L 1,3-PDO
after 60 h.
E. coli strains are able to produce 1,3-PDO from glycerol. In an earlier
example, Tong et al. (1991) constructed a 1,3-PDO-producing strain by
expression of dha system from K. pneumonia and the 0.46 mol/mol yield
of 1,3-PDO from glycerol was achieved. Similarly, Wang et al. (2007)
obtained a recombinant E. coli strain expressing a K. pneumonia glycerol
dehydratase, and a reactivating factor for the glycerol dehydratase and a
alcohol dehydrogenase from E. coli produced 13.2 g/L 1,3-PDO.
1,2-PDO is also an important bulk chemical mainly derived from
propylene. It is a useful material for the production of polyester, antifreeze, and de-icer. Although the bio-based route for 1,2-PDO production has been investigated more than 20 years, low titers and yields
were barriers for commercial application of this alternative route.
Altaras and Cameron (1999, 2000) constructed various recombinant
E. coli strains to produce 1,2-PDO from glucose by coexpression of
methylglyoxal synthase (coded by mgsA), glycerol dehydrogenase
(coded by gldA), and either yeast alcohol dehydrogenase or E. coli
1,2-PD oxidoreductase. Using a combination of elimination of
byproduct production and optimization of the anaerobic fed-batch
fermentation process, a titers up to 4.5 g/L, and yields up to 0.19 g/g
glucose were achieved. More recently, Clomburg and Gonzalez (2011)
developed an E. coli strain for production of 1,2-PDO from glycerol
based on their previously established platform for fermentative metabolism of glycerol. E. coli was genetically engineered by overexpression
of mgsA, gldA and yqhD genes, and substitution of the native E. coli
PEP-dependent dihydroxyacetone kinase (DHAK) with an ATPdependent DHAK from Citrobacter freundii. With the disruption of
undesirable by-product pathways producing acetate and lactate, the
resulting strain produced 5.6 g/L 1,2-PDO at a yield of 21.3% (g/g)
glycerol. Nonetheless, the titers and yields of 1,2-PDO are fairly low in
above examples which largely determine the competitiveness of the
biotechnological process and strain improvement needs further effort
to enhance conversion efciency of 1,2-PDO (Zeng and Sabra, 2011).
Compared to 1,4-BDO, 2,3-BDO can be naturally accumulated in

several microbial species such as Klebsiella and Bacillus but not
E. coli. 2,3-BDO exists in three stereoisomers forms: levo-D-()-,
dextro-L-(+)- and meso-form. Recently, microbial production of
2,3-BDO were reviewed in detail (Celinska and Grajek, 2009; Ji et al.,
2011). Therefore, we will discuss here only the recent progress in
metabolic engineering for E. coli to produce 2,3-BDO.
To develop an E. coli able to produce isomers of 2,3-BDO, three
enzymes involved in 2,3-BDO biosynthesis pathway were transferred
into the host from K. pneumonia and the achieved recombinant strain
produced up to 17.7 g/L meso-2,3-BDO from glucose (Ui et al., 1997).
Genetic engineering was also attempted to produce other isomers of
this compound. Ui et al. (2004) constructed an L-2,3-BDO producing
E. coli strain by simultaneous expression of diacetyl reductase from
K. pneumoniae and L-2,3-butanediol dehydrogenase from Brevibacterium
saccharolyticum. This E. coli strain generated 2.2 g/L L-2,3-BDO from 3 g/L
diacetyl which corresponded to a 73% conversion rate. However this
new recombinant strain had a possible problem with substrate inhibition and by-product formation of meso-2,3-BDO. Similarly, screening
and overexpression of the key enzymes for meso-2,3-BDO formation
from different organisms in E. coli host resulted in a titer up to 1.12 g/L
with a yield of 0.29 g/g glucose (Nielsen et al., 2010). Although the
by-products pathway was disrupted, the redox imbalance signicantly
impaired the fermentation performance of the recombinant cell.
Recently, a recombinant E. coli strain able to produce a higher titer
and higher purity (enantio purity >99%) of (R,R)-2,3-BDO production
was developed by Yan et al. (2009). To construct this organism, four
secondary alcohol dehydrogenases (sADHs) were characterized for
their activity and stereospecicity towards acetoin reduction, and
amplied in E. coli to enhance the 2,3-BDO synthetic pathway. These
manipulations achieved enantiomerically pure (R,R)-2,3-BDO with a
titer of 6.1 g/L and with a glucose yield of 0.31 g/g. In another interesting
example, E. coli strain was manipulated to collectively produce chiral
acetoin and pure (S,S)-2,3-BDO using a cofactor regenerating system.
The recombinant strains co-expressed the NAD-dependent (2R,3R)2,3-BD dehydrogenase, which catalyzes the stereospecic oxidation of
(2R,3R)-2,3-BDO and meso-2,3-BDO to (3R)-acetoin and (3S)-acetoin,
respectively, and an unique NADH oxidase from L. brevis, which regenerates NAD+ from NADH by reducing O2 to H2O. This strain generated the
chiral acetoin and pure (S,S)-2,3-BDO with high biocatalytic efciency
from the racemic mixture of 2,3-BDO (Xiao et al., 2010).
6.2. 1,4-Butanediol and 2,3-butanediol

1,4-Butanediol (1,4-BDO) and 2,3-butanediol (2,3-BDO), are
important C4 diols platform chemicals used to manufacture of polymers, cosmetics, ne chemicals and solvents. 2,3-BDO production can
be obtained either by the chemical route or by the biotechnological
route (Zeng and Sabra, 2011), whereas1,4-BDO production comes
exclusively from fossil fuel feed-stocks because of the apparent absence
of 1,4-BDO biosynthesis pathways in known organisms (Yim et al.,
2011). A biological process for 1,4-BDO production may has become a
major effort but many challenges remain.
A breakthrough of bio-production of 1,4-BDO from renewable
biomass-derived feedstocks by engineered E. coli was recently developed based on the advances of synthetic biology and systematic metabolic engineering (Yim et al., 2011). In this signicant work, 1,4-BDO
pathway was designed in E. coli by the manipulation of six reactions
catalyzed by two native enzymes and four heterologous enzymes
from succinic acid as a central metabolic intermediate to 1,4-BDO.
Codon optimization, elimination of pathways leading to byproducts,
and increasing available carbon and reducing equivalents was manipulated. The resulting strain produced 1,4-BDO from different sugars such
as glucose, sucrose, and xylose with a titer up to 18 g/L. It is believed
that this strategy of systemic-level metabolic engineering could be
applied to design other biocatalysts and industrially important targeted
chemicals.
6.3. Polyhydroxyalkanoates production

Polyhydroxyalkanoates (PHAs) are a group of biopolyesters that
are intracellularly accumulated by numerous microorganisms when
unfavorable growth conditions are emerged such as environmental
stress or nutrient limitation (Chen, 2009; Lee, 1996). Although their
excellent material properties and complete biodegradability are
promising substitutes for traditional plastics, it appears that high production costs are a major hindrance for a wide range of applications
(Ahn et al., 2000; Nikel et al., 2006). Recently, PHAs applications also
have been expanded in biofuel industry (Gao et al., 2011). To date,
much effort has been devoted to cost-effective production of PHAs
through strain development, and optimization of the fermentation
and recovery processes (Gao et al., 2011; Kang et al., 2008; Lee, 1996).
In particular, recombinant production of PHA through the establishment of biosynthesis pathways in non-polymer-producing heterologous hosts has proven to be an encouraging development (Park and
Lee, 2003, 2004; Park et al., 2012; Zhang et al., 1994). One example is
the large-scale commercial production of PHA by fermentation of
recombinant E. coli. Moreover, an E. coli strain was one of the most
frequent hosts for biopolymer production because of its advantages
such as a wide range of utilizable carbon sources, accumulation of a
large amount of polymers with a high level of productivity, high cell
density fermentation, and the fragility of cells (Choi and Lee, 1999;
Rehm, 2010).
In terms of versatile characteristics, PHAs can be classied into
three main types based on the sizes of the monomers: short-chain
length (scl) consisting of monomer units of C3 to C5, medium chain
length (mcl) consisting of monomer units of C6 to C14, and scl-mcl
PHAs consisting of monomers ranging in size from C4 to C14. Metabolic
engineering approaches have been employed to develop an E. coli strain
able to efciently convert cheap renewable feedstocks to PHSs
(Andreessen et al., 2010; Kang et al., 2004; Li et al., 2007; Nikel et al.,
2006), and to synthesize novel PHA homopolymers and copolymers
(Chen, 2009; Nomura et al., 2004; Park et al., 2012). Many research
groups have constructed recombinant E. coli strains to produce PHA, especially polyhydroxybutyrate (PHB, the most widespread and
best-known PHAs), by introducing genes responsible for PHAs biosynthesis into the host from different organisms such as Pseudomonas
aeruginosa (Langenbach et al., 1997; Qi et al., 1997), Alcaligenes latus
(Choi et al., 1998), Thiocapsa pfennigii (Liu and Steinbuchel, 2000) and
Azotobacter species (Nikel et al., 2006). In a notable example, constitutive expression of an operon responsible for PHA biosynthesis pathway
of A. latus in E. coli resulted in the accumulation PHB with a unprecedented titer of 140 g/L, and a high productivity of 4.63 g/L/h when a
pH-stat fed-batch process was used. This recombinant strain synthesized PHB more efciently than those of harboring the Ralstonia
eutropha genes (Choi et al., 1998) and has demonstrated a higher
productivity compared to those native PHAs producers.
Chen and coworkers developed a number of recombinant E. coli
strains for production of PHA homopolymers and copolymers including
PHB (Li et al., 2009), poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
[P(3HB-co-3HV)] (Jian et al., 2010), poly(3-hydroxybutyrate-co-4hydroxybutyrate) [P(3HB-co-4HB)] (Li et al., 2010), and poly(3hydroxybutyrate-co-3-hydroxyhexanoate) (Lu et al., 2003). Among
them, 23.5 g/L cell dry weight containing 62.7% P(3HB-co-4HB) with a
12.5 mol % 4HB monomer content was obtained from glucose, which
is the highest 4HB monomer content in P(3HB-co-4HB) produced
from unrelated carbon sources. In addition, coexpression of the mutant
fabH (encoding 3-ketoacyl-ACP synthase III) and phaC genes in E. coli
JM109 resulted in scl-mcl PHAs copolymer production from glucose
(Nomura et al., 2004). This study indicated that implementation of
metabolic engineering can control the composition of PHA copolymers.
Furthermore, the above mentioned recombinant E. coli strain harboring
PHA biosynthesis pathway of A. latus was also used to accumulate
P(3HB-co-3HV), which is more exible and stronger than PHB increasing
its range of applications. When an improved nutrient feeding strategy,
acetic acid induction, and oleic acid supplementation were applied,
203.1 g/L cell concentration and 158.8 g/L P(3HB-co-3HV) were
achieved with a 10.6 mol% 3HV fraction and a high productivity of
2.88 g/L/h (Choi and Lee, 1999). In addition, the metabolic engineering
strategy was designed to develop E. coli cells able to produce PHAs
containing 2HB monomer (Park et al., 2012).
Recombinant E. coli efciently converts inexpensive carbon
sources into a diverse range of PHAs with different chemical and
material properties, which is an important approach for lowering the
production cost. Therefore, the economic feasibility of PHAs production
is a crucial parameter in the evaluation of biocatalyst efciency. A
recombinant E. coli strain bearing the PHA biosynthetic genes from Azotobacter sp. was created to utilize whey together with corn steep liquor
as main carbon and nitrogen sources for PHA accumulation (Nikel et al.,
2006). Constitutive expression of genes involved in lactose transport
and utilization together with the absence of the lactose repressor generated efcient PHB production with an intracellular concentration
of 72.9% of the cell dry weight, and a volumetric productivity of
2.13 g/L/h in a fed-batch laboratory-scale bioreactor. In another study,
using a recombinant E. coli strain harboring the A. latus PHA biosynthesis genes, the pH-stat fed-batch cultures were carried out with a concentrated whey solution containing 280 g/L lactose to produce nal
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cell and PHB concentrations of 119.5 and 96.2 g/L, respectively, with a
productivity of 2.57 g/L/h (Ahn et al., 2000). With similar substrates,
Jae Park et al. (2002) investigated PHA production by recombinant
E. coli in a 30-and 300-L scale fermenters, and 35.5 g/L PHB with a
70% polymer content and 20 g/L PHB with a 67% polymer content
were obtained, respectively. Xylose and cellulose hydrolysates have
also been evaluated for PHB production by a genetically E. coli strain
(Lee, 1998). Recently, Metabolix, Inc. and Archer Daniel Midland
(ADM) have opened the rst commercial-scale plant to produce a
corn syrup-based PHB by recombinant E. coli (Chen, 2009).
6.4. Polylactic acid and copolymers production
Polylactic acid (PLA) and its copolymers are promising biomassderived polymers because of their merits such as biodegradability,
biocompatibility, compostability, and low toxicity to humans (Lasprilla
et al., 2012; Mehta et al., 2005). The typical PLA production route
contains two steps: the fermentative production of lactate and the
subsequently chemically ring-opening polymerization. At present the
commercial process is complicated and costly (Jung and Lee, 2011). An
exciting and recent development is the bio-based one-step production
of polylactic acid by construction of synthetic pathways in E. coli as
a cell factory (Jung et al., 2010; Yang et al., 2010). In these studies,
Clostridium propionicum propionate CoA-transferase and Pseudomonas
sp PHA synthase (PhaC) were subjected to directed enzyme evolution
to improve their bioconversion performance in the respective generation of lactyl-CoA and incorporation of lactyl-CoA into the polymer.
The genes encoding the engineered propionate-CoA transferase and
PhaC were introduced into E. coli host and thus in vivo biosynthetic pathways for the production of PLA and lactate-containing polymers were
established. Owing to the low titer of polymer, in silico genome-scale
metabolic-ux analysis of E. coli was applied to reveal further geneticengineering approaches. Based on the information of in silico genomescale simulation, the metabolic pathways were rationally engineered
by increasing additional precursors and knocking out the competing
pathways including ackA, ppc and adhE genes as well as replacing the
promoters of D-lactate dehydrogenase (ldhA) and acetyl-CoA synthetase (acs) genes with the strong trc promoter. Accordingly, PLA homopolymer production was notably enhanced up to 11% of dry cell
weight from glucose by this engineered strain.
More recently, this E. coli strain was further engineered to simplify
the fermentation process by overcoming induction expression and
feeding succinic acid, and consequently efcient production of PLA
and lactate-containing copolymers was achieved (Jung and Lee,
2011). In addition metabolic engineering of bacterial biosynthesis
pathways in an E. coli host has led to the heterologous production of
new unnatural polymers, such as polythioesters and lactate-based
polyesters (Lutke-Eversloh et al., 2002; Taguchi et al., 2008).
Homopolythioesters were produced from the precursor substrate,
3-mercaptoalkanoate by a recombinant E. coli strain harboring
genes encoding phosphotransbutyrylase and butyrate kinase from
Clostridium acetobutylicum and the nonspecic PHA synthase from
T. pfennigii. These new polymers showed the unique properties with
low crystalline order or improved thermal stability when compared
with PHAs and petrochemical-derived polymers.
7. Biosynthesis of complex natural compounds in E. coli
Secondary metabolites are a class of extremely structural-diverse
compounds, which are naturally synthesized in low quantities by
their native hosts but serve many critical functions in an organism's
survivability and reproduction (Mitchell, 2011; Pitera et al., 2007).
Furthermore, this class of natural products has been extensively
used in industrial, agricultural and biomedical production. However,
owing to sophisticated structure of these molecules, efcient production by the chemical route is difcult (Mijts and Schmidt-Dannert,
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2003). On the other hand, traditional methods of strain improvement

are limited to manipulating secondary products pathways because of
the complicated metabolic network and multiple-regulatory circuits.
Recent developments of metabolic engineering and synthetic biology
have resulted in tools to enable overproduction of target products or
biosynthesis of novel compounds which were previously impossible.,
Examples are isoprenoids and derivates (Ajikumar et al., 2010; Kim
et al., 2010; Pitera et al., 2007; Wang et al., 1999; Yuan et al., 2006),
polyketides (Pfeifer et al., 2001; Zhang et al., 2008), non-ribosomal
peptides (Gruenewald et al., 2004; Watanabe et al., 2006), coenzyme
Q10 (Kim et al., 2006; Zahiri et al., 2006b) as well as biopolymers and
monomers as discussed above. In this section, some successful examples of engineering E. coli to synthesize secondary metabolites will be
discussed concisely and efcient strategies will be highlighted.
7.1. Isoprenoid biosynthesis
Isoprenoid compounds, the most diverse class of natural products
containing monoterpenes, sesquiterpenes, diterpenes, and triterpenes
as well as carotenoids, have broad applications in avors, fragrances,
and medicines (Misawa, 2011). Two main metabolic routes are
known for biosynthesis of the isoprenoid precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the
mevalonate-dependent pathway (MVA) (in eukaryotes), which is catalytically accomplished by six enzymatic reactions from acetyl-coA, and
the mevalonate independent methylerythritol phosphate pathway
(MEP) (in E. coli, most other bacteria, and plants) (Fig. 5). In E. coli,
the MEP pathway comprises seven catalytic steps from an initial
condensation reaction between pyruvate and glyceraldehyde-3phosphate (G3P) catalyzed by DXP synthase (DXS) to form 1-deoxyD-xylulose-5-phosphate (DXP). After rearrangement of DXP, it undergoes reduction to MEP by DXP reductoisomerase (DXR) encoded
by ispC (dxr). Some MEP pathway enzymes encoded by ispD, ispE, ispF,
ispG, and ispH are then used in subsequent sequential reactions to
convert MEP into the building blocks of IPP and DMAPP, which are
isomerized via the enzyme encoded by idi (Fig. 5) (Hoefer et al.,
2002; Mijts and Schmidt-Dannert, 2003). In the MVA pathway the IPP
biosynthesis begins with the conversion of three molecules of
acetyl-CoA to MVA through acetoacetyl-CoA and HMG-CoA. MVA is
converted to MVA diphosphate by two phosphorylation reactions
mediated by MVA kinase and phospho-MVA kinase, respectively. Then
MVA diphosphate undergoes dehydrationdecarboxylation in an ATPrequiring reaction, resulting in IPP. An IPP isomerase catalyzes the isomerization of IPP to DMAPP (Fig. 5). Recent advances in this eld have
been reviewed thoroughly (Daum et al., 2009; Klein-Marcuschamer
et al., 2007; Misawa, 2011).
E. coli has been employed to overproduce the isoprenoid compounds via enhanced availability and abundance of precursors through
rational, random or combined approaches (Kajiwara et al., 1997; KleinMarcuschamer et al., 2007; Wang et al., 1999). In an important development, recombinant E. coli was employed to produce carotenoids, in
which native dxs gene was overexpressed to increase the isoprenoid
precursor pool and a non-native gene clusters responsible for carotenoid biosynthetic pathway was introduced from Erwinia uredovora
(Matthews and Wurtzel, 2000). Furthermore, Kim et al. (2010)
engineered the Brevibacterium linens carotenoid pathways in E. coli to
introduce more diverse molecular structure. Promiscuous carotenoidmodifying enzymes combined with the combinatorial biosynthesis (a
strategy of engineering the bacterial multienzyme polyketide synthases
to produce novel drug candidates by modular genetic architecture of
these enzymes) generated structurally diverse compounds that are
hardly accessible in nature. Their work demonstrated that the reconstruction and redesign of heterologous pathway in E. coli offers a promising strategy for the extension of secondary metabolite production. In a
recent example, taxadiene, the precursor of a potent anticancer drug
Taxol that originate in plants, was produced with signicantly increased
titers in E. coli by metabolic engineering (Ajikumar et al., 2010). Native

DXP pathway and heterologous terpenoid-forming pathway were
combined and optimized to balance the overall ux distribution and
maximize the taxadiene production with minimal accumulation of
indole, which negatively affected the taxadiene synthesis and cell growth.
Therefore, the multivariate-modular pathway engineering approach led
to taxadiene production with an enhanced titer of 1 g/L by recombinant
cells. Similarly, E. coli was employed to produce labdane-related
diterpene natural products through a modular metabolic engineering
system. Enhancement of isoprenoid precursors resulting from incorporation of a heterologous MEV pathway and/or enhancement of the
endogenous MEP pathway led up to 1000-fold improvement in
diterpene production (Morrone et al., 2010).
In another example, Leonard et al. (2010) developed a
levopimaradiene producing E. coli through protein engineering and
metabolic engineering. Manipulations of the terpenoid pathway by
overexpressing the limiting enzymes in the MEP pathway and introducing two evolved genes, responsible for levopimaradiene synthase
and geranylgeranyl diphosphate synthase from plants, respectively,
resulted in enhanced production of levopimaradiene. Notably, evolution of these enzymes by protein engineering signicantly increased
the catalytic efciency for biosynthesis of the target product. In addition, some other strategies such as promoter engineering (Alper et al.,
2005; Yuan et al., 2006), multi-dimensional gene target search
approach (Jin and Stephanopoulos, 2007), and inactivation of competing
pathways at the pyruvate and acetyl-CoA nodes (Vadali et al., 2005),
were also attempted to increase the production of isoprenoid compounds. Briey, these strategies used in above examples not only offer
a cost effective production of desired isoprenoid compounds with a
reasonable yield, but also should be useful for the production of other
intricately natural/non-natural products that are presently inaccessible.
7.2. Nonribosomal peptides and polyketides
Nonribosomal peptides (NRPs) and polyketides (PKs), two important
natural product classes, exhibit a wide variety of clinically biological activities, such as antibiotics, antitumor or antifungal drugs or as immunosuppressive agents (Strieker et al., 2010; Watanabe et al., 2006;
Watanabe and Oikawa, 2007). Erythromycin, vancomycin and epothilone
represent just a few of these important natural compounds (Pfeifer et al.,
2003). Genes responsible for biosynthesis of such metabolites in bacteria
are generally assembled into clusters of polycistronic operons on the
chromosomes of the native host. In spite of broad therapeutic applications, these secondary metabolites are insufciently produced due to limitations of the original microbial hosts including low production levels,
poor growth and culture difculties, or rudimentary knowledge associated with biosynthesis network and regulatory circuits (Pfeifer et al., 2003;
Watanabe et al., 2009). Harnessing impressive advances in systems
metabolic approaches and synthetic biology, E. coli has successfully
been developed to produce these complex natural compounds by a number of research groups (Challis, 2006; Kreutzer et al., 2011; Watanabe and
Oikawa, 2007). Some elegant examples involved heterologous NRPs and
PKs production using E. coli as an efcient platform will be introduced
and useful strategies will be discussed here.
Generally, NRPs synthetases and PKs synthetases exist in gene
clusters composed of an assembly of distinct modular sections. Taking
account of the large size or number of genes expressed, in most cases
several plasmids containing mutually compatible origins of replication
were used to assemble the reconstituted partial gene clusters. The
example of the total biosynthesis of 6-deoxyerythronolide B (6dEB) in
a genetically engineered E. coli strain represented a breakthrough for
engineered biosynthesis of bacterial complex natural polyketides
(Pfeifer et al., 2001). The multiple manipulations with the introduction
of the three deoxyerythronolide B synthase genes from Saccharopolyspora
erythraea, sfp gene from B. subtilis and the heterodimeric propionyl-CoA
carboxylase genes from Streptomyces coelicolor, combined the deletion
OP
Pyruvate
1217
Glyceraldehyde
3-phosphate
Acetyl-CoA
Acetyl-CoA
COA
COA
dxs
AAS
OH
1-Deoxylulose-5-phosphate
Acetoacetyl-CoA
OP
O
COA
OH
HMGS
dxr (ispC)
OH
OH
4-Cytidine-5'-diphospho2-methylerythritol
Hydroxymethylglutaryl
-CoA
HOOC
COA
OP
OH
OH
HMGR
ispD
OH
OH
2-Methylerythritol4-phosphate
OH
HOOC
OPP-cyt
OH
Mevalonate
MK
OH
OH
ispE
Mevalonate-5-phosphate
OP
OP
2-Phospho-4-cytidine-5'diphospho-2-methylerythritol
HOOC
PMK
OPP-cyt
OH
OH
Mevalonatediphosphate
OH
OPP
ispF
HOOC
OP
PMD
PO
Methyleryhtritol-2,4cyclodiphosphate
OH
MVA pathway
OH
ispG
1-Hydroxy-2-methyl-2OPP butenyl-4-diphosphate
OH
ispH
MEP pathway
OPP
idi
Dimethylallyldiphosphate
OPP
Isopentanyldiphosphate
Fig. 5. Mevalonate and non-mevalonate isoprenoid pathways in E. coli strain (Daum et al., 2009; Misawa, 2011). Red boxes indicate the genes encoding the corresponding key
enzymes and are shown: dxs, 1-deoxy-D-xylulose 5-phosphate synthase; dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; ispD, 4-diphosphocytidyl-2-methylery-thritol
synthase; ispE, 4-diphosphocytidyl-2-methylerythritol kinase; ispF, 2-methylerythritol-2,4-cyclodiphosphate synthase; ispG, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate
synthase; ispH, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase; idi, isopentenyl diphosphate-dimethylallyl diphosphate isomerase; AAS, acetoacetyl-CoA synthase;
HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; PMD, mevalonate diphosphatede carboxylase.
of the endogenous prpRBCD genes as well as overexpression of the endogenous prpE and birA genes were achieved. When gene expression was
coordinately induced at low temperature, the resulting cellular catalyst
produced 6dEB from propionate with excellent kinetic parameters. In

another example, the initial steps in the biosynthesis of the decapeptide
antibiotic tyocidine A were reconstituted in E. coli on the basis of an
1218
engineered NRPS system (Gruenewald et al., 2004). The cyclic dipeptide

D-Phe-Pro-diketopiperazine (D-Phe-Pro-DKP) pathway was enhanced
by expression of the bimodular NRPS system tycA/tycB1 from B. brevis
ATCC8185 with a reassembled form polycistronic operon and stable
chromosomal integration of the phosphopantetheine transferase
(which is required for posttranslational modication of nonribosomal
peptide synthetases into the active holoform) gene sfp (Gruenewald
et al., 2004). Comprehensive optimization of parameters affecting the
productivity of desired product led to produce 9.2 mg/L D-Phe-Pro-DKP.
Similarly, multicistronic expression plasmids harboring the yersiniabactin
synthetase enzyme complex which contains two large, modular proteins
were introduced into E. coli host and enabled heterologously synthesis of
a polyketide-nonribosomal peptide hybrid natural product yersiniabactin
(Miller et al., 2002; Pfeifer et al., 2003). Final titers of about 67 mg/L were
reached using a high-cell-density fed-batch fermentation (Pfeifer et al.,
2003). Recently, Zhang et al. (2008) described an approach for the total
biosynthesis of bacterial aromatic polyketides of anthraquinone in E. coli
by using a dissected and reassembled fungal polyketide synthase. Highcell density fermentation resulted in desired product synthesis with a
titer of 3 mg/L.
With a different strategy, Watanabe et al. (2006) established an
E. coli-based plasmid-borne system for de novo production of the
antitumor drug NRP echinomycin. To reconstruct the entire echinomycin
biosynthetic pathway, 16 essential genes of which 15 genes isolated
from Streptomyces lasaliensis and sfp phosphopantetheinyl transferase
isolated from B. subtilis, were assembled into three separate plasmids
in a monocistronic form and transformed into E. coli. This
multimonocistronic arrangement with orthogonal origins of replication
and antibiotic resistance genes not only ensured the stable retention of
all three plasmids but also facilitated functional expression of each
gene in a controllable manner. The resulting strain produced 0.3 mg/L
echinomycin by fed-batch fermentation. Although their work provided
a novel technology for heterologous expression of biosynthetic gene
clusters in E. coli, the relatively low titer demonstrated that much more
effort is needed before such technology can be applied routinely for the
large-scale production of complex natural products (Challis, 2006).
7.3. Coenzyme Q10 production
Coenzyme Q10 (CoQ10) is the main coenzyme Q species, which is
a naturally occurring coenzyme formed from the conjugation of a
benzoquinone ring with a hydrophobic isoprenoid chain of varying
chain length. This compound provides therapeutic benets in several
human diseases such as cardiomyopathy, Parkinson's and Alzheimer's
disease (Cluis et al., 2007). In addition, CoQ10 is also used in the food
and cosmetic industries owing to its antioxidant properties. To meet the
growing demand for this class of compound, an E. coli heterologous production system which is better suited for commercial production compared to natural microbial producers such as Agrobacterium tumefaciens
and Paracoccus denitricans (Cluis et al., 2007) has been developed by
implementation of modern metabolic engineering.
CoQ10 biosynthesis pathway in recombinant E. coli is typically
composed of three parts: a) the synthesis of a quinonoid ring, b) the
synthesis of decaprenyl diphosphate, and c) quinonoid ring modication (Jeya et al., 2010). Formation of 4-hydroxybenzoate (pHBA) from
chorismate, which belongs to an intermediate metabolite generated
from the shikimate pathway as mentioned in Sections 4.3 and 4.4 and
shown in Fig. 4, is the rst step in the biosynthesis of CoQ and catalyzed
by chorismate pyruvate-lyase encoded by the ubiC gene. In the second
part, one of critical steps is the formation of decaprenyl diphosphate
(DPP) from farnesyl diphosphate (FPP) precursor by expression of
an exogenous decaprenyl diphosphate synthase (DPS), instead of
octaprenyl diphosphate (OPP) produced by wild-type E. coli. Based on
above multiple reactions, ring modications including prenylation,
hydroxylation, methylation and decarboxylation generate the desired
compounds. Thus, three strategies have been explored to engineer
E. coli for enhanced CoQ10 production: a) screening high specic DPS

towards DPP, b) increasing ux of the isoprenoid precursors, and
c) overexpressing selected ubi genes involved in the CoQ10 biosynthetic
pathway (Cluis et al., 2007; Zahiri et al., 2006b).
Heterologous expression of highly specic DPS towards DPP in
E. coli was carried out to produce high pure CoQ10 and decrease
undesired CoQ species. In E. coli, the main CoQ form present is
CoQ8, depending on an octaprenyl diphosphate synthase catalyzing
the formation of octaprenyl diphosphate (OPP). Therefore, it is necessary for CoQ10 synthesis to introduce a DPS from a CoQ10-producing
host. However, DPSs enzymes from various hosts led to accumulation
of CoQ10 and varying levels of CoQ8 and CoQ9 (Park et al., 2005;
Zahiri et al., 2006a). From scale up point of view, impurity not only
results in complex purication processing in downstream but also
translates into increased product costs. Although DPS enzymes probably
generate shorter prenyl diphosphates in addition to DPP, Rhodobacter
sphaeroides DPS displayed higher towards DPP than that of from
A. tumefaciens (Zahiri et al., 2006a). Hence constructing overproducing CoQ10 E. coli by isolating and expressing DPS enzymes with
stringent product specicity might be an efcient approach to increase
yields and reduce the costly purication of CoQ10 from unwanted CoQ
species during the industrial manufacturing process.
The ux through the chorismate and MEP pathways are limiting in
E. coli, whereas both of them are requisite precursors for CoQ10
synthesis, as described in Sections 4.3 and 7.1. Therefore, to achieve
maximum yields of CoQ10 in recombinant host, an efcient approach
will be to increase the ux of both of these precursors. Disturbance of
the ux was employed by coexpression of the rate-limiting enzymes or
introduction of a heterologous pathway. For example, the overexpression of a MEP pathway rate-limiting enzyme DXS from
P. aeruginosa resulted in increases in CoQ10 content (Kim et al., 2006).
This recombinant E. coli produced CoQ10 with a maximum concentration of 46.1 mg/L from a glucose-limited fed-batch cultivation but the
titer was much lower than that of natural high producers. More recently,
Choi et al. (2009) have reported that knockout of the ispB gene and
expression of the A. tumefaciens dps gene in E. coli not only reduced
the accumulation of the unwanted CoQ species but improved the
CoQ10 production. In a successful example, Zahiri et al. (2006b)
reconstructed a complete foreign MVA pathway by introducing six
genes of Streptococcus pneumoniae as well as acetoactyl-CoA thiolase
from R. eutropha and IPP isomerase from E. coli to bypass the endogenous MEP pathway. The resulting recombinant E. coli produced 2.4 mg
CoQ10/g-dcw under the optimized culture conditions. The higher
CoQ10 content achieved so far in E. coli is indeed comparable to that
from natural high producers. In addition, their studies demonstrated
that increasing the availability of the pHBA precursor also resulted in
to some extent enhanced CoQ10 production, indicating that pHBA availability is indeed limiting CoQ10 synthesis in the recombinant E. coli. In
other studies, overexpression of selected UBI enzymes that participate
in the downstream pathway from the isoprenoid and chorismate of
biosynthesis of CoQ in E. coli demonstrated an effective approach
for enhanced CoQ10 contents. Furthermore, amplication pHBA
prenyltransferase UbiA and/or chorismate lyase UbiC generated a significant impact in improving CoQ10 production (Zhang et al., 2007a).
8. Conclusion and future directions
Unquestionably, E. coli has long been the workhorse of molecular
biology and a preferred industrial host organism for genetic and
metabolic engineering applications. The scientic and technological
progress of industrial biotechnology has been estimated to make a
signicant contribution to promotion of green or sustainable chemistry.
The three key advantages of biotechnological process over traditional
chemical synthesis are the environmental friendliness, availability and
abundance of renewable feedstock, and selectivity and diversity of
desired products.
However, even with the fast and substantial developments of

biotechnology, microbial catalysts including E. coli host are not as
malleable as those in synthetic organic chemistry. For example,
many existing large volume industrial chemicals are produced only
at very low levels or are not produced at all naturally by microorganisms. So far, metabolic engineers must consider many limitations in
the development of microbial catalysts. Therefore, to realize the industrial potential of E. coli as a sustainable platform and address both global
and local concerns, considerable effort should be focused on the following points in future: (i) using simple, available and inexpensive starting
materials, (ii) identifying and eliminating bottlenecks in pathways
for the desired products, (iii) constructing robust biocatalysts, and
(iv) optimizing regulatory networks to maximize yields, titers and
productivity. In spite of many challenges, metabolic engineering has
been successfully applied in many instances, and with continued developments more applications will be possible. An intriguing development
of metabolic engineering has emerged in term of systems metabolic
engineering, in which systems biology, synthetic biology, and evolutionary engineering are combined and integrated. Moreover, the system
metabolic engineering approaches are becoming increasingly powerful
tools in developing E. coli strains for the production of chemicals and
materials such as polylactic acid, L-threonine and succinic acid.
On the other hand, advances in genome sequencing, functional
genomics, genome engineering and omics have had a substantial
impact on our understanding of biosynthesis pathways and discovery
of novel pathways and metabolites, and new functions of enzymes in
relevant organisms (Posfai et al., 2006; Wang et al., 2009). These biotechnological approaches have been applied to pathway reconstruction
and engineering towards improved production and designer cells
that are tailor-made for the desired chemical and process (Lee et al.,
2011b). Furthermore, recent developments of genetic techniques
make feasible signicant improvements in the tness and robustness
of biocatalysts such as high resistance to inhibitors (Wang et al.,
2011c), and increasing tolerance to environmental stress including
toxic chemicals, high temperatures, high osmolarity, or acidic pH conditions which commonly occur during industrial processing (Portnoy et
al., 2011; Reyes et al., 2011; Sandoval et al., 2011). In particular, global
transcription machinery engineering and adaptive laboratory evolution
have proved successful strategies to aid metabolic engineering for
enhanced cellular traits in E. coli and yeasts (Alper et al., 2006;
Portnoy et al., 2011). Presumably, their potential will be further
implemented with a promising future in developing, optimizing, and
deploying metabolically engineered E. coli for the production of fuels,
as well as commodity and specialty chemicals.
From an industrial perspective on the production of bio-based
chemicals, all of these technical advances combined with downstream
processing provide the E. coli organism with exibility and plasticity
essential for the engineering of an efcient biocatalytic system. Taking
advantage of E. coli as host and advances in metabolic engineering, the
bacterium as a sustainable biocatalyst will soon be a dominant and
preferred platform for various industrial applications and processes.
This now opens the door for bio-based catalysis that reduces our dependency on petroleum-derived chemical products and leads the way to a
truly bio-sustainable economy.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China No. 21006039 and the Sino-South Africa Cooperation Program
2009DFA31300.
References
Abe F, Horikoshi K. Enhanced production of isoamyl alcohol and isoamyl acetate by
ubiquitination-decient Saccharomyces cerevisiae mutants. Cell Mol Biol Lett
2005;10:3838.
1219
Ahn WS, Park SJ, Lee SY. Production of Poly(3-hydroxybutyrate) by fed-batch culture
of recombinant Escherichia coli with a highly concentrated whey solution.
Appl Environ Microbiol 2000;66:36247.
Ajikumar PK, Xiao WH, Tyo KE, Wang Y, Simeon F, Leonard E, et al. Isoprenoid pathway
optimization for taxol precursor overproduction in Escherichia coli. Science
2010;330:704.
Akinterinwa O, Cirino PC. Anaerobic obligatory xylitol production in Escherichia coli
strains devoid of native fermentation pathways. Appl Environ Microbiol 2011;77:
7069.
Akinterinwa O, Khankal R, Cirino PC. Metabolic engineering for bioproduction of sugar
alcohols. Curr Opin Biotechnol 2008;19:4617.
Alper H, Fischer C, Nevoigt E, Stephanopoulos G. Tuning genetic control through
promoter engineering. Proc Natl Acad Sci USA 2005;102:12678.
Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G. Engineering yeast transcription
machinery for improved ethanol tolerance and production. Science 2006;314:
15658.
Altaras NE, Cameron DC. Metabolic engineering of a 1,2-propanediol pathway in
Escherichia coli. Appl Environ Microbiol 1999;65:11805.
Altaras NE, Cameron DC. Enhanced production of (R)-1,2-propanediol by metabolically
engineered Escherichia coli. Biotechnol Prog 2000;16:9406.
Alterthum F, Ingram LO. Efcient ethanol production from glucose, lactose, and xylose
by recombinant Escherichia coli. Appl Environ Microbiol 1989;55:19438.
Andreessen B, Lange AB, Robenek H, Steinbuchel A. Conversion of glycerol to
poly(3-hydroxypropionate) in recombinant Escherichia coli. Appl Environ Microbiol
2010;76:6226.
Antoni D, Zverlov VV, Schwarz WH. Biofuels from microbes. Appl Microbiol Biotechnol
2007;77:2335.
Atsumi S, Liao JC. Directed evolution of Methanococcus jannaschii citramalate synthase
for biosynthesis of 1-propanol and 1-butanol by Escherichia coli. Appl Environ
Microbiol 2008a;74:78028.
Atsumi S, Liao JC. Metabolic engineering for advanced biofuels production from
Escherichia coli. Curr Opin Biotechnol 2008b;19:4149.
Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, et al. Metabolic
engineering of Escherichia coli for 1-butanol production. Metab Eng 2008a;10:
30511.
Atsumi S, Hanai T, Liao JC. Non-fermentative pathways for synthesis of branched-chain
higher alcohols as biofuels. Nature 2008b;451:869.
Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC. Engineering the isobutanol
biosynthetic pathway in Escherichia coli by comparison of three aldehyde
reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol 2010;85:6517.
Baez-Viveros JL, Osuna J, Hernandez-Chavez G, Soberon X, Bolivar F, Gosset G. Metabolic
engineering and protein directed evolution increase the yield of L-phenylalanine
synthesized from glucose in Escherichia coli. Biotechnol Bioeng 2004;87:51624.
Baez-Viveros JL, Flores N, Juarez K, Castillo-Espana P, Bolivar F, Gosset G. Metabolic
transcription analysis of engineered Escherichia coli strains that overproduce
L-phenylalanine. Microb Cell Fact 2007;6:30.
Balderas-Hernandez VE, Sabido-Ramos A, Silva P, Cabrera-Valladares N, HernandezChavez G, Baez-Viveros JL, et al. Metabolic engineering for improving anthranilate
synthesis from glucose in Escherichia coli. Microb Cell Fact 2009;8:19.
Bastian S, Liu X, Meyerowitz JT, Snow CD, Chen MM, Arnold FH. Engineered ketol-acid
reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan1-ol production at theoretical yield in Escherichia coli. Metab Eng 2011;13:34552.
Blankschien MD, Clomburg JM, Gonzalez R. Metabolic engineering of Escherichia coli for
the production of succinate from glycerol. Metab Eng 2010;12:40919.
Blattner FR, Plunkett 3rd G, Bloch CA, Perna NT, Burland V, Riley M, et al. The complete
genome sequence of Escherichia coli K-12. Science 1997;277:145362.
Blombach B, Schreiner ME, Holtko J, Bartek T, Oldiges M, Eikmanns BJ. L-valine
production with pyruvate dehydrogenase complex-decient Corynebacterium
glutamicum. Appl Environ Microbiol 2007;73:207984.
Blombach B, Schreiner ME, Bartek T, Oldiges M, Eikmanns BJ. Corynebacterium
glutamicum tailored for high-yield L-valine production. Appl Microbiol Biotechnol
2008;79:4719.
Bloor AE, Cranenburgh RM. An efcient method of selectable marker gene excision by
Xer recombination for gene replacement in bacterial chromosomes. Appl Environ
Microbiol 2006;72:25205.
Bond-Watts BB, Bellerose RJ, Chang MC. Enzyme mechanism as a kinetic control
element for designing synthetic biofuel pathways. Nat Chem Biol 2011;7:2227.
Booth IR, Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low KB, et al. Glycerol and
methylglyoxal metabolism. Escherichia coli and Salmonella: cellular and molecular
biology. Washington DC: ASM Press; 2005.
Bozell JJ, Petersen GR. Technology development for the production of biobased
products from biorenery carbohydratesthe US Department of Energy s Top 10
revisited. Green Chem 2010;12:53954.
Bruschi F, Dundar M, Gahan P, Gartland K, Szente M, Viola-Magni M, et al. Biotechnology
worldwide and the European Biotechnology Thematic Network Association
(EBTNA). Curr Opin Biotechnol 2011;22S:S7-S14.
Cai G, Jin B, Monis P, Saint C. Metabolic ux network and analysis of fermentative
hydrogen production. Biotechnol Adv 2011;29:37587.
Cann AF, Liao JC. Production of 2-methyl-1-butanol in engineered Escherichia coli. Appl
Microbiol Biotechnol 2008;81:8998.
Causey TB, Zhou S, Shanmugam KT, Ingram LO. Engineering the metabolism of
Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized
products: homoacetate production. Proc Natl Acad Sci USA 2003;100:82532.
Causey TB, Shanmugam KT, Yomano LP, Ingram LO. Engineering Escherichia coli for
efcient conversion of glucose to pyruvate. Proc Natl Acad Sci USA 2004;101:
223540.
1220
Celinska E, Grajek W. Biotechnological production of 2,3-butanediolcurrent state and

prospects. Biotechnol Adv 2009;27:71525.
Challis GL. Engineering Escherichia coli to produce nonribosomal peptide antibiotics.
Nat Chem Biol 2006;2:398400.
Chang DE, Jung HC, Rhee JS, Pan JG. Homofermentative production of D- or L-lactate in
metabolically engineered Escherichia coli RR1. Appl Environ Microbiol 1999;65:13849.
Chatterjee R, Millard CS, Champion K, Clark DP, Donnelly MI. Mutation of the ptsG gene
results in increased production of succinate in fermentation of glucose by
Chavez-Bejar MI, Lara AR, Lopez H, Hernandez-Chavez G, Martinez A, Ramirez OT, et al.
Metabolic engineering of Escherichia coli for L-tyrosine production by expression of
genes coding for the chorismate mutase domain of the native chorismate
mutase-prephenate dehydratase and a cyclohexadienyl dehydrogenase from
Zymomonas mobilis. Appl Environ Microbiol 2008;74:328490.
Chen GQ. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry.
Chem Soc Rev 2009;38:243446.
Chin JW, Cirino PC. Improved NADPH supply for xylitol production by engineered
Escherichia coli with glycolytic mutations. Biotechnol Prog 2011;27:33341.
Chin JW, Khankal R, Monroe CA, Maranas CD, Cirino PC. Analysis of NADPH supply
during xylitol production by engineered Escherichia coli. Biotechnol Bioeng
2009;102:20920.
Choi JI, Lee SY. High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
by fed-batch culture of recombinant Escherichia coli. Appl Environ Microbiol
1999;65:43638.
Choi JI, Lee SY, Han K. Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis
genes and use of these genes for enhanced production of poly(3-hydroxybutyrate) in
Choi JH, Ryu YW, Park YC, Seo JH. Synergistic effects of chromosomal ispB deletion and
dxs overexpression on coenzyme Q(10) production in recombinant Escherichia coli
expressing Agrobacterium tumefaciens dps gene. J Biotechnol 2009;144:649.
Cirino PC, Chin JW, Ingram LO. Engineering Escherichia coli for xylitol production from
glucosexylose mixtures. Biotechnol Bioeng 2006;95:116776.
Clomburg JM, Gonzalez R. Biofuel production in Escherichia coli: the role of metabolic
engineering and synthetic biology. Appl Microbiol Biotechnol 2010;86:41934.
Clomburg JM, Gonzalez R. Metabolic engineering of Escherichia coli for the production
of 1,2-propanediol from glycerol. Biotechnol Bioeng 2011;108:86779.
Cluis CP, Burja AM, Martin VJ. Current prospects for the production of coenzyme Q10 in
microbes. Trends Biotechnol 2007;25:51421.
Connor MR, Liao JC. Engineering of an Escherichia coli strain for the production of
3-methyl-1-butanol. Appl Environ Microbiol 2008;74:576975.
Connor MR, Cann AF, Liao JC. 3-Methyl-1-butanol production in Escherichia coli:
random mutagenesis and two-phase fermentation. Appl Microbiol Biotechnol
2010;86:115564.
Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia
coli K-12 using PCR products. Proc Natl Acad Sci USA 2000;97:66405.
Daum M, Herrmann S, Wilkinson B, Bechthold A. Genes and enzymes involved in
bacterial isoprenoid biosynthesis. Curr Opin Chem Biol 2009;13:1808.
Delhomme C, Weuster-Botz D, Kuhn FE. Succinic acid from renewable resources as a C4
building-block chemicala review of the catalytic possibilities in aqueous media.
Green Chem 2008;11:1326.
Dharmadi Y, Murarka A, Gonzalez R. Anaerobic fermentation of glycerol by Escherichia
coli: a new platform for metabolic engineering. Biotechnol Bioeng 2006;94:8219.
Dien BS, Nichols NN, Bothast RJ. Fermentation of sugar mixtures using Escherichia coli
catabolite repression mutants engineered for production of L-lactic acid. J Ind
Dong X, Quinn PJ, Wang X. Metabolic engineering of Escherichia coli and Corynebacterium
glutamicum for the production of L-threonine. Biotechnol Adv 2011;29:1123.
Edwards MC, Henriksen EDC, Yomano LP, Gardner BC, Sharma LN, Ingram LO, et al.
Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for
fuel ethanol production from pectin-rich lignocellulosic biomass. Appl Environ
Microbiol 2011;77(15):518491.
Eiteman MA, Lee SA, Altman E. A co-fermentation strategy to consume sugar mixtures
effectively. J Biol Eng 2008;2:3.
Eiteman MA, Lee SA, Altman R, Altman E. A substrate-selective co-fermentation strategy
with Escherichia coli produces lactate by simultaneously consuming xylose and
glucose. Biotechnol Bioeng 2009;102:8227.
Elisakova V, Patek M, Holatko J, Nesvera J, Leyval D, Goergen JL, et al. Feedback-resistant
acetohydroxy acid synthase increases valine production in Corynebacterium
glutamicum. Appl Environ Microbiol 2005;71:20713.
Emptage M, Haynie SL, Laffend LA, Pucci JP, Whited G. Process for the biological production of 1, 3-propanediol with high titer. US: Patent 2003; 6514733.
Escalante A, Calderon R, Valdivia A, de Anda R, Hernandez G, Ramirez OT, et al.
Metabolic engineering for the production of shikimic acid in an evolved Escherichia
coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase
system. Microb Cell Fact 2010;9:21.
Flores N, Xiao J, Berry A, Bolivar F, Valle F. Pathway engineering for the production of
aromatic compounds in Escherichia coli. Nat Biotechnol 1996;14:6203.
Furukawa S, Ozaki A, Kotani Y, Nakanishi T. Breeding of L-threonine hyper-producer of
Escherichia coli W. Appl Microbiol Biotechnol 1988;29:2537.
Gao X, Chen J-C, Wu Q, Chen G-Q. Polyhydroxyalkanoates as a source of chemicals,
polymers, and biofuels. Curr Opin Biotechnol 2011;22:76874.
Geddes CC, Mullinnix MT, Nieves IU, Peterson JJ, Hoffman RW, York SW, et al. Simplied
process for ethanol production from sugarcane bagasse using hydrolysate-resistant
Escherichia coli strain MM160. Bioresour Technol 2011a;102:270211.
Geddes CC, Nieves IU, Ingram LO. Advances in ethanol production. Curr Opin
Biotechnol 2011b;22:3129.
Gerigk M, Bujnicki R, Ganpo-Nkwenkwa E, Bongaerts J, Sprenger G, Takors R. Process

control for enhanced L-phenylalanine production using different recombinant
Escherichia coli strains. Biotechnol Bioeng 2002;80:74654.
Gonzalez R, Murarka A, Dharmadi Y, Yazdani SS. A new model for the anaerobic
fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in
Escherichia coli. Metab Eng 2008;10:23445.
Gruenewald S, Mootz HD, Stehmeier P, Stachelhaus T. In vivo production of articial
nonribosomal peptide products in the heterologous host Escherichia coli.
Hanai T, Atsumi S, Liao JC. Engineered synthetic pathway for isopropanol production in
Heuser F, Marin K, Kaup B, Bringer S, Sahm H. Improving D-mannitol productivity of
Escherichia coli: impact of NAD, CO2 and expression of a putative sugar permease
from Leuconostoc pseudomesenteroides. Metab Eng 2009;11:17883.
Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, et al. Biomass
recalcitrance: engineering plants and enzymes for biofuels production. Science
2007;315:8047.
Hoefer JF, Hemmerlin A, Grosdemange-Billiard C, Bach TJ, Rohmer M. Isoprenoid
biosynthesis in higher plants and in Escherichia coli: on the branching in the
methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl
diphosphate and dimethylallyl diphosphate. Biochem J 2002;366:57383.
Hoffmann P. Tomorrow's energy hydrogen, fuel cells, and the prospects for a cleaner
planet. London: MIT Press; 2002.
Hong SH, Lee SY. Importance of redox balance on the production of succinic acid by
metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 2002;58:
28690.
Huffer S, Roche CM, Blanch HW, Clark DS. Escherichia coli for biofuel production: bridging
the gap from promise to practice. Trends Biotechnol 2012;30:53845.
Ikeda M. Amino acid production processes. Adv Biochem Eng Biotechnol 2003;79:1-35.
Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF. Genetic engineering of ethanol
production in Escherichia coli. Appl Environ Microbiol 1987;53:24205.
Inokuma K, Liao JC, Okamoto M, Hanai T. Improvement of isopropanol production by
metabolically engineered Escherichia coli using gas stripping. J Biosci Bioeng
2010;110:696701.
Jantama K, Haupt MJ, Svoronos SA, Zhang X, Moore JC, Shanmugam KT, et al. Combining
metabolic engineering and metabolic evolution to develop nonrecombinant strains
of Escherichia coli C that produce succinate and malate. Biotechnol Bioeng 2008a;99:
114053.
Jantama K, Zhang X, Moore JC, Shanmugam KT, Svoronos SA, Ingram LO. Eliminating
side products and increasing succinate yields in engineered strains of Escherichia
coli C. Biotechnol Bioeng 2008b;101:88193.
Jeya M, Moon HJ, Lee JL, Kim IW, Lee JK. Current state of coenzyme Q(10) production
and its applications. Appl Microbiol Biotechnol 2010;85:165363.
Ji XJ, Huang H, Ouyang PK. Microbial 2,3-butanediol production: a state-of-the-art
review. Biotechnol Adv 2011;29:35164.
Jian J, Zhang SQ, Shi ZY, Wang W, Chen GQ, Wu Q. Production of polyhydroxyalkanoates
by Escherichia coli mutants with defected mixed acid fermentation pathways. Appl
Jiang M, Liu SW, Ma JF, Chen KQ, Yu L, Yue FF, et al. Effect of growth phase feeding
strategies on succinate production by metabolically engineered Escherichia coli.
Jin LH, Lee JH. Change in proteomic proles of genetically modied 1,3-propanediolproducing recombinant E. coli. J Microbiol Biotechnol 2008;18:143944.
Jin YS, Stephanopoulos G. Multi-dimensional gene target search for improving lycopene
biosynthesis in Escherichia coli. Metab Eng 2007;9:33747.
Johansson L, Lindskog A, Silfversparre G, Cimander C, Nielsen KF, Liden G. Shikimic acid
production by a modied strain of E. coli (W3110.shik1) under phosphate-limited
and carbon-limited conditions. Biotechnol Bioeng 2005;92:54152.
Jojima T, Inui M, Yukawa H. Production of isopropanol by metabolically engineered
Escherichia coli. Appl Microbiol Biotechnol 2008;77:121924.
Jones DT, Woods DR. Acetonebutanol fermentation revisited. Microbiol Rev 1986;50:
484524.
Jun SA, Moon C, Kang CH, Kong SW, Sang BI, Um Y. Microbial fed-batch production of
1,3-propanediol using raw glycerol with suspended and immobilized Klebsiella
pneumoniae. Appl Biochem Biotechnol 2010;161:491501.
Jung YK, Lee SY. Efcient production of polylactic acid and its copolymers by metabolically
engineered Escherichia coli. J Biotechnol 2011;151:94-101.
Jung YK, Kim TY, Park SJ, Lee SY. Metabolic engineering of Escherichia coli for the
production of polylactic acid and its copolymers. Biotechnol Bioeng 2010;105:16171.
Kadam KL, Chin CY, Brown LW. Flexible biorenery for producing fermentation sugars,
lignin and pulp from corn stover. J Ind Microbiol Biotechnol 2008;35:33141.
Kajiwara S, Fraser PD, Kondo K, Misawa N. Expression of an exogenous isopentenyl
diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli.
Biochem J 1997;324(Pt. 2):4216.
Kang Z, Wang Q, Zhang H, Qi Q. Construction of a stress-induced system in Escherichia
coli for efcient polyhydroxyalkanoates production. Appl Microbiol Biotechnol
2008;79:2038.
Kang Z, Du L, Kang J, Wang Y, Wang Q, Liang Q, et al. Production of succinate and
polyhydroxyalkanoate from substrate mixture by metabolically engineered
Escherichia coli. Bioresour Technol 2011;102:66004.
Kaup B, Bringer-Meyer S, Sahm H. Metabolic engineering of Escherichia coli: construction
of an efcient biocatalyst for D-mannitol formation in a whole-cell biotransformation.
Appl Microbiol Biotechnol 2004;64:3339.
Kedar P, Colah R, Shimizu K. Proteomic investigation on the pyk-F gene knockout
Escherichia coli for aromatic amino acid production. Enzyme Microb Technol
2007;41:45565.

Khamduang M, Packdibamrung K, Chutmanop J, Chisti Y, Srinophakun P. Production of
L-phenylalanine from glycerol by a recombinant Escherichia coli. J Ind Microbiol
Biotechnol 2009;36:126774.
Khankal R, Chin JW, Cirino PC. Role of xylose transporters in xylitol production from
engineered Escherichia coli. J Biotechnol 2008;134:24652.
Kim MS, Lee DY. Fermentative hydrogen production from tofuprocessing waste
and anaerobic digester sludge using microbial consortium. Bioresour Technol
2010;101(Suppl. 1):S4852.
Kim P, Laivenieks M, Vieille C, Zeikus JG. Effect of overexpression of Actinobacillus
succinogenes phosphoenolpyruvate carboxykinase on succinate production in
Kim SJ, Kim MD, Choi JH, Kim SY, Ryu YW, Seo JH. Amplication of 1-deoxy-D-xyluose
5-phosphate (DXP) synthase level increases coenzyme Q10 production in
recombinant Escherichia coli. Appl Microbiol Biotechnol 2006;72:9825.
Kim S, Seol E, Oh YK, Wang G, Park S. Hydrogen production and metabolic ux analysis
of metabolically engineered Escherichia coli strains. Int J Hydrogen Energy 2009;34:
741727.
Kim SH, Park YH, Schmidt-Dannert C, Lee PC. Redesign, reconstruction, and directed
extension of the Brevibacterium linens C40 carotenoid pathway in Escherichia coli.
Klein-Marcuschamer D, Ajikumar PK, Stephanopoulos G. Engineering microbial cell
factories for biosynthesis of isoprenoid molecules: beyond lycopene. Trends
Biotechnol 2007;25:41724.
Ko BS, Rhee CH, Kim JH. Enhancement of xylitol productivity and yield using a xylitol
dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic
conditions. Biotechnol Lett 2006;28:115962.
Kreutzer MF, Kage H, Gebhardt P, Wackler B, Saluz HP, Hoffmeister D, et al. Biosynthesis of
a complex yersiniabactin-like natural product via the mic locus in phytopathogen
Ralstonia solanacearum. Appl Environ Microbiol 2011;77:611724.
Kromer JO, Wittmann C, Schroder H, Heinzle E. Metabolic pathway analysis for rational
design of L-methionine production by Escherichia coli and Corynebacterium
glutamicum. Metab Eng 2006;8:35369.
Kumar R, Shimizu K. Transcriptional regulation of main metabolic pathways of cyoA,
cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic
continuous cultures. Microb Cell Fact 2011;10:3.
Langenbach S, Rehm BH, Steinbuchel A. Functional expression of the PHA synthase
gene phaC1 from Pseudomonas aeruginosa in Escherichia coli results in poly(3hydroxyalkanoate) synthesis. FEMS Microbiol Lett 1997;150:3039.
Lasprilla AJ, Martinez GA, Lunelli BH, Jardini AL, Filho RM. Poly-lactic acid synthesis for
application in biomedical devicesa review. Biotechnol Adv 2012;30:3218.
Lee SY. Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production
in bacteria. Trends Biotechnol 1996;14:4318.
Lee SY. Poly (3-hydroxybutyrate) production from xylose by recombinant Escherichia
coli. Bioprocess Biosyst Eng 1998;18:3979.
Lee YJ, Cho JY. Genetic manipulation of a primary metabolic pathway for L-ornithine
production in Escherichia coli. Biotechnol Lett 2006;28:184956.
Lee SY, Hong SH, Moon SY. In silico metabolic pathway analysis and design: succinic acid
production by metabolically engineered Escherichia coli as an example. Genome
Inform 2002;13:21423.
Lee JH, Lee DE, Lee BU, Kim HS. Global analyses of transcriptomes and proteomes of a
parent strain and an L-threonine-overproducing mutant strain. J Bacteriol
2003a;185:544251.
Lee JK, Song JY, Kim SY. Controlling substrate concentration in fed-batch Candida
magnoliae culture increases mannitol production. Biotechnol Prog 2003b;19:
76875.
Lee M, Smith GM, Eiteman MA, Altman E. Aerobic production of alanine by Escherichia
coli aceF ldhA mutants expressing the Bacillus sphaericus alaD gene. Appl Microbiol
Lee SJ, Lee DY, Kim TY, Kim BH, Lee J, Lee SY. Metabolic engineering of Escherichia coli
for enhanced production of succinic acid, based on genome comparison and
in silico gene knockout simulation. Appl Environ Microbiol 2005;71:78807.
Lee SJ, Song H, Lee SY. Genome-based metabolic engineering of Mannheimia
succiniciproducens for succinic acid production. Appl Environ Microbiol 2006;72:
193948.
Lee KH, Park JH, Kim TY, Kim HU, Lee SY. Systems metabolic engineering of Escherichia
coli for L-threonine production. Mol Syst Biol 2007;3:149.
Lee JH, Sung BH, Kim MS, Blattner FR, Yoon BH, Kim JH, et al. Metabolic engineering of a
reduced-genome strain of Escherichia coli for L-threonine production. Microb Cell
Fact 2009;8:2.
Lee JW, Kim HU, Choi S, Yi J, Lee SY. Microbial production of building block chemicals
and polymers. Curr Opin Biotechnol 2011a;22:75867.
Lee JY, Yang KS, Jang SA, Sung BH, Kim SC. Engineering butanol-tolerance in Escherichia
coli with articial transcription factor libraries. Biotechnol Bioeng 2011b;108:
7429.
Leonard E, Ajikumar PK, Thayer K, Xiao WH, Mo JD, Tidor B, et al. Combining metabolic
and protein engineering of a terpenoid biosynthetic pathway for overproduction
and selectivity control. Proc Natl Acad Sci USA 2010;107:136549.
Li R, Chen Q, Wang PG, Qi Q. A novel-designed Escherichia coli for the production of
various polyhydroxyalkanoates from inexpensive substrate mixture. Appl Microbiol
Li ZJ, Cai L, Wu Q, Chen GQ. Overexpression of NAD kinase in recombinant Escherichia
coli harboring the phbCAB operon improves poly (3-hydroxybutyrate) production.
Li ZJ, Shi ZY, Jian J, Guo YY, Wu Q, Chen GQ. Production of poly(3-hydroxybutyrateco-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered
Escherichia coli. Metab Eng 2010;12:3529.
1221
Lin H, Bennett GN, San KY. Fed-batch culture of a metabolically engineered Escherichia
coli strain designed for high-level succinate production and yield under aerobic
conditions. Biotechnol Bioeng 2005a;90:7759.
Lin H, Bennett GN, San KY. Genetic reconstruction of the aerobic central metabolism in
Escherichia coli for the absolute aerobic production of succinate. Biotechnol Bioeng
2005b;89:14856.
Lin H, Bennett GN, San KY. Metabolic engineering of aerobic succinate production
systems in Escherichia coli to improve process productivity and achieve the maximum
theoretical succinate yield. Metab Eng 2005c;7:11627.
Liu SJ, Steinbuchel A. A novel genetically engineered pathway for synthesis of
poly(hydroxyalkanoic acids) in Escherichia coli. Appl Environ Microbiol 2000;66:
73943.
Lu X, Zhang J, Wu Q, Chen GQ. Enhanced production of poly(3-hydroxybutyrateco-3-hydroxyhexanoate) via manipulating the fatty acid beta-oxidation pathway
in E. coli. FEMS Microbiol Lett 2003;221:97-101.
Lu S, Eiteman MA, Altman E. Effect of CO2 on succinate production in dual-phase
Escherichia coli fermentations. J Biotechnol 2009;143:21323.
Lutke-Eversloh T, Fischer A, Remminghorst U, Kawada J, Marchessault RH,
Bogershausen A, et al. Biosynthesis of novel thermoplastic polythioesters by
engineered Escherichia coli. Nat Mater 2002;1:23640.
Maeda T, Sanchez-Torres V, Wood TK. Metabolic engineering to enhance bacterial
hydrogen production. Microb Biotechnol 2008;1:309.
Matthews PD, Wurtzel ET. Metabolic engineering of carotenoid accumulation in
Escherichia coli by modulation of the isoprenoid precursor pool with expression
of deoxyxylulose phosphate synthase. Appl Microbiol Biotechnol 2000;53:396400.
Mazumdar S, Clomburg JM, Gonzalez R. Escherichia coli strains engineered for
homofermentative production of D-lactic acid from glycerol. Appl Environ
Microbiol 2010;76:432736.
Mehta R, Kumar V, Bhunia H, Upadhyay S. Synthesis of poly (lactic acid): a review.
J Macromol Sci Part C: Polymer Rev 2005;45:32549.
Mijts BN, Schmidt-Dannert C. Engineering of secondary metabolite pathways. Curr
Opin Biotechnol 2003;14:597602.
Millard CS, Chao YP, Liao JC, Donnelly MI. Enhanced production of succinic acid by
overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. Appl Environ
Microbiol 1996;62:180810.
Miller DA, Luo L, Hillson N, Keating TA, Walsh CT. Yersiniabactin synthetase: a
four-protein assembly line producing the nonribosomal peptide/polyketide hybrid
siderophore of Yersinia pestis. Chem Biol 2002;9:33344.
Miller EN, Jarboe LR, Turner PC, Pharkya P, Yomano LP, York SW, et al. Furfural inhibits
growth by limiting sulfur assimilation in ethanologenic Escherichia coli strain
LY180. Appl Environ Microbiol 2009a;75:613241.
Miller EN, Jarboe LR, Yomano LP, York SW, Shanmugam KT, Ingram LO. Silencing of
NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant
ethanologenic Escherichia coli. Appl Environ Microbiol 2009b;75:431523.
Mills TY, Sandoval NR, Gill RT. Cellulosic hydrolysate toxicity and tolerance mechanisms in
Escherichia coli. Biotechnol Biofuels 2009;2:26.
Misawa N. Pathway engineering for functional isoprenoids. Curr Opin Biotechnol
2011;22:62733.
Mitchell W. Natural products from synthetic biology. Curr Opin Chem Biol 2011;15:
50515.
Mohan Raj S, Rathnasingh C, Jung WC, Park S. Effect of process parameters on
3-hydroxypropionic acid production from glycerol using a recombinant Escherichia
coli. Appl Microbiol Biotechnol 2009;84:64957.
Moniruzzaman M, Lai X, York SW, Ingram LO. Isolation and molecular characterization
of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic
Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon. Appl Environ
Microbiol 1997;63:46337.
Moon TS, Yoon SH, Lanza AM, Roy-Mayhew JD, Prather KL. Production of glucaric acid
from a synthetic pathway in recombinant Escherichia coli. Appl Environ Microbiol
2009;75:58995.
Moon TS, Dueber JE, Shiue E, Prather KL. Use of modular, synthetic scaffolds for improved
production of glucaric acid in engineered E. coli. Metab Eng 2010;12:298305.
Morrone D, Lowry L, Determan MK, Hershey DM, Xu M, Peters RJ. Increasing diterpene
yield with a modular metabolic engineering system in E. coli: comparison of MEV
and MEP isoprenoid precursor pathway engineering. Appl Microbiol Biotechnol
2010;85:1893906.
Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R. Fermentative utilization of glycerol by
Escherichia coli and its implications for the production of fuels and chemicals. Appl
Environ Microbiol 2008;74:112435.
Murphy KC, Campellone KG, Poteete AR. PCR-mediated gene replacement in
Escherichia coli. Gene 2000;246:32130.
Nakamura CE, Whited GM. Metabolic engineering for the microbial production of
1,3-propanediol. Curr Opin Biotechnol 2003;14:4549.
Nakamura CE, Gatenby AA, Hsu AKH, La Reau RD, Haynie SL, Diaz-Torres M, et al. Method
for the production of 1, 3-propanediol by recombinant microorganisms. US Patent;
2000; 6013494.
Narayanan N, Roychoudhury PK, Srivastava A. L (+) lactic acid fermentation and its
product polymerization. J Biotechnol 2004;7:16778.
Nielsen DR, Yoon SH, Yuan CJ, Prather KL. Metabolic engineering of acetoin and meso-2,
3-butanediol biosynthesis in E. coli. Biotechnol J 2010;5:27484.
Nieves IU, Geddes CC, Mullinnix MT, Hoffman RW, Tong Z, Castro E, et al. Injection of air
into the headspace improves fermentation of phosphoric acid pretreated sugarcane
bagasse by Escherichia coli MM170. Bioresour Technol 2011;102:695965.
Nikel PI, de Almeida A, Melillo EC, Galvagno MA, Pettinari MJ. New recombinant
Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate)
from agroindustrial by-products. Appl Environ Microbiol 2006;72:394954.
1222
Nikel PI, Giordano AM, de Almeida A, Godoy MS, Pettinari MJ. Elimination of D-lactate
synthesis increases poly(3-hydroxybutyrate) and ethanol synthesis from glycerol
and affects cofactor distribution in recombinant Escherichia coli. Appl Environ
Microbiol 2010;76:74006.
Nomura CT, Taguchi K, Taguchi S, Doi Y. Coexpression of genetically engineered
3-ketoacyl-ACP synthase III (fabH) and polyhydroxyalkanoate synthase (phaC)
genes leads to short-chain-length-medium-chain-length polyhydroxyalkanoate
copolymer production from glucose in Escherichia coli JM109. Appl Environ
Microbiol 2004;70:999-1007.
Oh YK, Raj SM, Jung GY, Park S. Current status of the metabolic engineering of microorganisms for biohydrogen production. Bioresour Technol 2011;102:835767.
Ohta K, Alterthum F, Ingram LO. Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli. Appl Environ Microbiol 1990;56:4635.
Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO. Genetic improvement of
Escherichia coli for ethanol production: chromosomal integration of Zymomonas
mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.
Okano K, Tanaka T, Ogino C, Fukuda H, Kondo A. Biotechnological production of
enantiomeric pure lactic acid from renewable resources: recent achievements,
perspectives, and limits. Appl Microbiol Biotechnol 2010;85:41323.
Olson MM, Templeton LJ, Suh W, Youderian P, Sariaslani FS, Gatenby AA, et al. Production of
tyrosine from sucrose or glucose achieved by rapid genetic changes to phenylalanineproducing Escherichia coli strains. Appl Microbiol Biotechnol 2007;74:103140.
Panagiotopoulos IA, Bakker RR, Budde MA, de Vrije T, Claassen PA, Koukios EG. Fermentative
hydrogen production from pretreated biomass: a comparative study. Bioresour Technol
2009;100:63318.
Park SJ, Lee SY. Identication and characterization of a new enoyl coenzyme A
hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates
in recombinant Escherichia coli. J Bacteriol 2003;185:53917.
Park SJ, Lee SY. New FadB homologous enzymes and their use in enhanced biosynthesis
of medium-chain-length polyhydroxyalkanoates in FadB mutant Escherichia coli.
Biotechnol Bioeng 2004;86:6816.
Park JH, Lee SY. Towards systems metabolic engineering of microorganisms for amino
acid production. Curr Opin Biotechnol 2008;19:45460.
Park SJ, Park JP, Lee SY. Production of poly (3-hydroxybutyrate) from whey by
fed-batch culture of recombinant Escherichia coli in a pilot-scale fermenter.
Biotechnol Lett 2002;24:1859.
Park YC, Kim SJ, Choi JH, Lee WH, Park KM, Kawamukai M, et al. Batch and fed-batch
production of coenzyme Q10 in recombinant Escherichia coli containing the
decaprenyl diphosphate synthase gene from Gluconobacter suboxydans. Appl
Park JH, Lee KH, Kim TY, Lee SY. Metabolic engineering of Escherichia coli for the
production of L-valine based on transcriptome analysis and in silico gene knockout
simulation. Proc Natl Acad Sci U S A 2007;104:7797802.
Park JH, Jang YS, Lee JW, Lee SY. Escherichia coli W as a new platform strain for the
enhanced production of L-valine by systems metabolic engineering. Biotechnol
Bioeng 2011;108:11407.
Park SJ, Lee TW, Lim SC, Kim TW, Lee H, Kim MK, et al. Biosynthesis of polyhydroxyalkanoates
containing 2-hydroxybutyrate from unrelated carbon source by metabolically
engineered Escherichia coli. Appl Microbiol Biotechnol 2012;93:27383.
Pfeifer BA, Admiraal SJ, Gramajo H, Cane DE, Khosla C. Biosynthesis of complex
polyketides in a metabolically engineered strain of E. coli. Science 2001;291:17902.
Pfeifer BA, Wang CC, Walsh CT, Khosla C. Biosynthesis of Yersiniabactin, a complex
polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host.
Pitera DJ, Paddon CJ, Newman JD, Keasling JD. Balancing a heterologous mevalonate
pathway for improved isoprenoid production in Escherichia coli. Metab Eng
2007;9:193207.
Plachy J. The effect of medium composition on the production of valine by Corynebacterium 9366EMS/184. Folia Microbiol (Praha) 1975;20:34650.
Polen T, Kramer M, Bongaerts J, Wubbolts M, Wendisch VF. The global gene expression
response of Escherichia coli to L-phenylalanine. J Biotechnol 2005;115:22137.
Portnoy VA, Bezdan D, Zengler K. Adaptive laboratory evolutionharnessing the power
of biology for metabolic engineering. Curr Opin Biotechnol 2011;22:5904.
Posfai G, Plunkett III G, Feher T, Frisch D, Keil GM, Umenhoffer K, et al. Emergent
properties of reduced-genome Escherichia coli. Science 2006;312:10446.
Qi Q, Rehm BH, Steinbuchel A. Synthesis of poly(3-hydroxyalkanoates) in Escherichia
coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa:
comparison of PhaC1 and PhaC2. FEMS Microbiol Lett 1997;157:15562.
Rathnasingh C, Raj SM, Jo JE, Park S. Development and evaluation of efcient recombinant
Escherichia coli strains for the production of 3-hydroxypropionic acid from glycerol.
Rehm BHA. Bacterial polymers: biosynthesis, modications and applications. Nat Rev
Microbiol 2010;8:57892.
Reyes LH, Almario MP, Kao KC. Genomic library screens for genes involved in n-butanol
tolerance in Escherichia coli. PLoS One 2011;6:e17678.
Sakakibara Y, Saha BC, Taylor P. Microbial production of xylitol from L-arabinose by
metabolically engineered Escherichia coli. J Biosci Bioeng 2009;107:50611.
Sanchez AM, Bennett GN, San KY. Efcient succinic acid production from glucose through
overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase
and lactate dehydrogenase mutant. Biotechnol Prog 2005;21:35865.
Sandoval NR, Mills TY, Zhang M, Gill RT. Elucidating acetate tolerance in E. coli using a
genome-wide approach. Metab Eng 2011;13:21424.
Sanny T, Arnaldos M, Kunkel SA, Pagilla KR, Stark BC. Engineering of ethanolic E. coli
with the Vitreoscilla hemoglobin gene enhances ethanol production from both
glucose and xylose. Appl Microbiol Biotechnol 2010;88:110312.
Sauer M, Porro D, Mattanovich D, Branduardi P. Microbial production of organic acids:

expanding the markets. Trends Biotechnol 2008;26:1008.
Savrasova EA, Kivero AD, Shakulov RS, Stoynova NV. Use of the valine biosynthetic pathway to convert glucose into isobutanol. J Ind Microbiol Biotechnol 2011;38:128794.
Schaub J, Mauch K, Reuss M. Metabolic ux analysis in Escherichia coli by integrating
isotopic dynamic and isotopic stationary 13C labeling data. Biotechnol Bioeng
2008;99:117085.
Seo MY, Seo JW, Heo SY, Baek JO, Rairakhwada D, Oh BR, et al. Elimination of
by-product formation during production of 1,3-propanediol in Klebsiella pneumoniae
by inactivation of glycerol oxidative pathway. Appl Microbiol Biotechnol 2009;84:
52734.
Seol E, Manimaran A, Jang Y, Kim S, Oh YK, Park S. Sustained hydrogen production from
formate using immobilized recombinant Escherichia coli SH5. Int J Hydrogen Energy
2011;36:86816.
Shams Yazdani S, Gonzalez R. Engineering Escherichia coli for the efcient conversion of
glycerol to ethanol and co-products. Metab Eng 2008;10:34051.
Shen CR, Liao JC. Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol
production via the keto-acid pathways. Metab Eng 2008;10:31220.
Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC. Driving forces enable high-titer
anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol 2011;77:
290515.
Shiio I, Nakamori S. Microbial production of L-threonine. Part I. Produciton by
Escherichia coli mutant resistant to a -amino--hydroxyvaleric acid. Agric Biol
Chem 1969;33:115260.
Singh A, Cher Soh K, Hatzimanikatis V, Gill RT. Manipulating redox and ATP balancing
for improved production of succinate in E. coli. Metab Eng 2011;13:7681.
Sonderegger M, Sauer U. Evolutionary engineering of Saccharomyces cerevisiae for
anaerobic growth on xylose. Appl Environ Microbiol 2003;69:19908.
Song KH, Lee HH, Hyun HH. Characterization of salt-tolerant mutant for enhancement
of L-threonine production in Escherichia coli. Appl Microbiol Biotechnol 2000;54:
64751.
Sprenger GA. Aromatic amino acids. Amino acid biosynthesis pathways, regulation and
metabolic engineering. In: Steinbchel A, editor. Microbiology Monographs. Berlin:
Springer; 2007a.
Sprenger GA. From scratch to value: engineering Escherichia coli wild type cells to the
production of L-phenylalanine and other ne chemicals derived from chorismate.
Appl Microbiol Biotechnol 2007b;75:73949.
Stols L, Donnelly MI. Production of succinic acid through overexpression of NAD+dependent malic enzyme in an Escherichia coli mutant. Appl Environ Microbiol
1997;63:2695701.
Strieker M, Tanovic A, Marahiel MA. Nonribosomal peptide synthetases: structures and
dynamics. Curr Opin Struct Biol 2010;20:23440.
Sun JF, Xu M, Zhang F, Wang ZX. Novel recombinant E. coli producing ethanol from
glucose and xylose. Acta Microbiol Sin (China) 2004;44:6004.
Suzuki T, Yokoyama S, Kinoshita Y, Yamada H, Hatsu M, Takamizawa K, et al. Expression of
xyrA gene encoding for D-xylose reductase of Candida tropicalis and production of
xylitol in Escherichia coli. J Biosci Bioeng 1999;87:2804.
Taguchi S, Yamada M, Matsumoto K, Tajima K, Satoh Y, Munekata M, et al. A microbial
factory for lactate-based polyesters using a lactate-polymerizing enzyme. Proc Natl
Acad Sci USA 2008;105:173237.
Takors R, Bathe B, Rieping M, Hans S, Kelle R, Huthmacher K. Systems biology for industrial
strains and fermentation processesexample: amino acids. J Biotechnol 2007;129:
18190.
Toivari MH, Ruohonen L, Miasnikov AN, Richard P, Penttila M. Metabolic engineering of
Saccharomyces cerevisiae for conversion of D-glucose to xylitol and other vecarbon sugars and sugar alcohols. Appl Environ Microbiol 2007;73:54716.
Tomar A, Eiteman MA, Altman E. The effect of acetate pathway mutations on the
production of pyruvate in Escherichia coli. Appl Microbiol Biotechnol 2003;62:
7682.
Tong IT, Liao HH, Cameron DC. 1,3-Propanediol production by Escherichia coli expressing
genes from the Klebsiella pneumoniae dha regulon. Appl Environ Microbiol 1991;57:
35416.
Trinh CT, Srienc F. Metabolic engineering of Escherichia coli for efcient conversion of
glycerol to ethanol. Appl Environ Microbiol 2009;75:6696705.
Trinh CT, Unrean P, Srienc F. Minimal Escherichia coli cell for the most efcient production
of ethanol from hexoses and pentoses. Appl Environ Microbiol 2008;74:363443.
Turner PC, Miller EN, Jarboe LR, Baggett CL, Shanmugam KT, Ingram LO. YqhC regulates
transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved
in furfural tolerance. J Ind Microbiol Biotechnol 2011;38:4319.
Ui S, Okajima Y, Mimura A, Kanai H, Kudo T. Molecular generation of an Escherichia coli
strain producing only the meso-isomer of 2, 3-butanediol. J Ferment Bioeng
1997;84:1859.
Ui S, Takusagawa Y, Sato T, Ohtsuki T, Mimura A, Ohkuma M, et al. Production of
L-2,3-butanediol by a new pathway constructed in Escherichia coli. Lett Appl
Microbiol 2004;39:5337.
Vadali RV, Fu Y, Bennett GN, San KY. Enhanced lycopene productivity by manipulation
of carbon ow to isopentenyl diphosphate in Escherichia coli. Biotechnol Prog
2005;21:155861.
Vemuri GN, Eiteman MA, Altman E. Succinate production in dual-phase Escherichia
coli fermentations depends on the time of transition from aerobic to anaerobic
conditions. J Ind Microbiol Biotechnol 2002;28:32532.
Wada M, Narita K, Yokota A. Alanine production in an H+-ATPase- and lactate
dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase.
Wang CW, Oh MK, Liao JC. Engineered isoprenoid pathway enhances astaxanthin
production in Escherichia coli. Biotechnol Bioeng 1999;62:23541.

Wang Q, Chen X, Yang Y, Zhao X. Genome-scale in silico aided metabolic analysis
and ux comparisons of Escherichia coli to improve succinate production. Appl
Wang F, Qu H, Zhang D, Tian P, Tan T. Production of 1,3-propanediol from glycerol by
recombinant E. coli using incompatible plasmids system. Mol Biotechnol 2007;37:
1129.
Wang Z, Chen M, Xu Y, Li S, Lu W, Ping S, et al. An ethanol-tolerant recombinant
Escherichia coli expressing Zymomonas mobilis pdc and adhB genes for enhanced
ethanol production from xylose. Biotechnol Lett 2008;30:65763.
Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, et al. Programming cells by
multiplex genome engineering and accelerated evolution. Nature 2009;460:8948.
Wang J, Zhu J, Bennett GN, San KY. Succinate production from different carbon sources
under anaerobic conditions by metabolic engineered Escherichia coli strains. Metab
Eng 2011a;13:32835.
Wang J, Zhu J, Bennett GN, San KY. Succinate production from sucrose by metabolic
engineered Escherichia coli strains under aerobic conditions. Biotechnol Prog
2011b;27:12427.
Wang X, Miller EN, Yomano LP, Zhang X, Shanmugam KT, Ingram LO. Increased furfural
tolerance due to overexpression of NADH-dependent oxidoreductase FucO in
Escherichia coli strains engineered for the production of ethanol and lactate. Appl
Environ Microbiol 2011c;77:513240.
Wang X, Miller EN, Yomano LP, Shanmugam KT, Ingram LO. Increased furan tolerance
in Escherichia coli due to a cryptic ucpA gene. Appl Environ Microbiol 2012;78:
24525.
Watanabe K, Oikawa H. Robust platform for de novo production of heterologous
polyketides and nonribosomal peptides in Escherichia coli. Org Biomol Chem
2007;5:593602.
Watanabe K, Hotta K, Praseuth AP, Koketsu K, Migita A, Boddy CN, et al. Total biosynthesis
of antitumor nonribosomal peptides in Escherichia coli. Nat Chem Biol 2006;2:4238.
Watanabe K, Praseuth AP, Praseuth MB, Hotta K. Chapter 15. Plasmid-borne gene cluster
assemblage and heterologous biosynthesis of nonribosomal peptides in Escherichia
coli. Methods Enzymol 2009;458:37999.
Wee Y, Kim J, Ryu H. Biotechnological production of lactic acid and its recent applications.
Food Technol Biotechnol 2006;44:16372.
Wendisch VF, Bott M, Eikmanns BJ. Metabolic engineering of Escherichia coli and
Corynebacterium glutamicum for biotechnological production of organic acids and
amino acids. Curr Opin Microbiol 2006;9:26874.
Weusthuis RA, Lamot I, van der Oost J, Sanders JP. Microbial production of bulk
chemicals: development of anaerobic processes. Trends Biotechnol 2011;9:1538.
Wu H, Li ZM, Zhou L, Ye Q. Improved succinic acid production in the anaerobic culture
of an Escherichia coli pB ldhA double mutant as a result of enhanced anaplerotic
activities in the preceding aerobic culture. Appl Environ Microbiol 2007;73:
783743.
Xiao Z, Lv C, Gao C, Qin J, Ma C, Liu Z, et al. A novel whole-cell biocatalyst with NAD+
regeneration for production of chiral chemicals. PLoS One 2010;5:e8860.
Xu YZ, Guo NN, Zheng ZM, Ou XJ, Liu HJ, Liu DH. Metabolism in 1,3-propanediol
fed-batch fermentation by a D-lactate decient mutant of Klebsiella pneumoniae.
Yan Y, Lee CC, Liao JC. Enantioselective synthesis of pure (R, R)-2,3-butanediol in
Escherichia coli with stereospecic secondary alcohol dehydrogenases. Org Biomol
Chem 2009;7:39147.
Yang TH, Kim TW, Kang HO, Lee SH, Lee EJ, Lim SC, et al. Biosynthesis of polylactic acid
and its copolymers using evolved propionate CoA transferase and PHA synthase.
Yi J, Li K, Draths KM, Frost JW. Modulation of phosphoenolpyruvate synthase expression
increases shikimate pathway product yields in E. coli. Biotechnol Prog 2002;18:
11418.
Yim H, Haselbeck R, Niu W, Pujol-Baxley C, Burgard A, Boldt J, et al. Metabolic engineering
of Escherichia coli for direct production of 1,4-butanediol. Nat Chem Biol 2011;7:
44552.
Yomano LP, York SW, Shanmugam KT, Ingram LO. Deletion of methylglyoxal synthase
gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli.
Yu C, Cao Y, Zou H, Xian M. Metabolic engineering of Escherichia coli for biotechnological
production of high-value organic acids and alcohols. Appl Microbiol Biotechnol
2011;89:57383.
1223
Yuan LZ, Rouviere PE, Larossa RA, Suh W. Chromosomal promoter replacement of the
isoprenoid pathway for enhancing carotenoid production in E. coli. Metab Eng
2006;8:7990.
Zahiri HS, Noghabi KA, Shin YC. Biochemical characterization of the decaprenyl diphosphate
synthase of Rhodobacter sphaeroides for coenzyme Q10 production. Appl Microbiol
Biotechnol 2006a;73:796806.
Zahiri HS, Yoon SH, Keasling JD, Lee SH, Won Kim S, Yoon SC, et al. Coenzyme Q10
production in recombinant Escherichia coli strains engineered with a heterologous
decaprenyl diphosphate synthase gene and foreign mevalonate pathway. Metab
Eng 2006b;8:40616.
Zaldivar J, Martinez A, Ingram LO. Effect of alcohol compounds found in hemicellulose
hydrolysate on the growth and fermentation of ethanologenic Escherichia coli.
Zelic B, Gostovic S, Vuorilehto K, Vasic-Racki D, Takors R. Process strategies to enhance
pyruvate production with recombinant Escherichia coli: from repetitive fed-batch
to in situ product recovery with fully integrated electrodialysis. Biotechnol Bioeng
2004;85:63846.
Zeng AP, Sabra W. Microbial production of diols as platform chemicals: recent progresses.
Curr Opin Biotechnol 2011;22:74951.
Zhang H, Obias V, Gonyer K, Dennis D. Production of polyhydroxyalkanoates in
sucrose-utilizing recombinant Escherichia coli and Klebsiella strains. Appl Environ
Microbiol 1994;60:1198205.
Zhang D, Shrestha B, Li Z, Tan T. Ubiquinone-10 production using Agrobacterium
tumefaciens dps gene in Escherichia coli by coexpression system. Mol Biotechnol
2007a;35:1-14.
Zhang X, Jantama K, Moore JC, Shanmugam KT, Ingram LO. Production of L-alanine by
metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 2007b;77:
35566.
Zhang W, Li Y, Tang Y. Engineered biosynthesis of bacterial aromatic polyketides in
Escherichia coli. Proc Natl Acad Sci USA 2008;105:206838.
Zhang X, Jantama K, Moore JC, Jarboe LR, Shanmugam KT, Ingram LO. Metabolic evolution
of energy-conserving pathways for succinate production in Escherichia coli. Proc Natl
Acad Sci USA 2009;106:201805.
Zhang X, Shanmugam KT, Ingram LO. Fermentation of glycerol to succinate by metabolically
engineered strains of Escherichia coli. Appl Environ Microbiol 2010;76:2397401.
Zhang F, Rodriguez S, Keasling JD. Metabolic engineering of microbial pathways for
advanced biofuels production. Curr Opin Biotechnol 2011;22:77583.
Zhao ZJ, Zou C, Zhu YX, Dai J, Chen S, Wu D, et al. Development of L-tryptophan production
strains by dened genetic modication in Escherichia coli. J Ind Microbiol Biotechnol
2011;38:19219.
Zheng H, Wang X, Yomano LP, Shanmugam KT, Ingram LO. Increase in furfural tolerance
in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA.
Zhou S, Causey TB, Hasona A, Shanmugam KT, Ingram LO. Production of optically pure
D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli
W3110. Appl Environ Microbiol 2003;69:399407.
Zhou S, Yomano LP, Shanmugam KT, Ingram LO. Fermentation of 10% (w/v) sugar to D:
()-lactate by engineered Escherichia coli B. Biotechnol Lett 2005;27:18916.
Zhou S, Shanmugam KT, Yomano LP, Grabar TB, Ingram LO. Fermentation of 12% (w/v)
glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium.
Zhou H, Liao X, Liu L, Wang T, Du G, Chen J. Enhanced L-phenylalanine production by
recombinant Escherichia coli BR-42 (pAP-B03) resistant to bacteriophage BP-1 via
a two-stage feeding approach. J Ind Microbiol Biotechnol 2011a;38:121927.
Zhou L, Zuo ZR, Chen XZ, Niu DD, Tian KM, Prior BA, et al. Evaluation of genetic manipulation
strategies on D-lactate production by Escherichia coli. Curr Microbiol 2011b;62:9819.
Zhou L, Tian KM, Niu DD, Shen W, Shi GY, Singh S, et al. Improvement of D-lactate
productivity in recombinant Escherichia coli by coupling production with growth.
Biotechnol Lett 2012a;34:112330.
Zhou L, Niu DD, Tian KM, Chen XZ, Prior BA, Shen W, et al. Genetically switched D-lactate
production in Escherichia coli. Metab Eng 2012b;14:5608.
Zhu Y, Eiteman M, DeWitt K, Altman E. Homolactate fermentation by metabolically
engineered Escherichia coli strains. Appl Environ Microbiol 2007;73:45664.
Zhu Y, Eiteman MA, Altman R, Altman E. High glycolytic ux improves pyruvate
production by a metabolically engineered Escherichia coli strain. Appl Environ
Microbiol 2008;74:664955.

Metabolic Engineering of E. Coli. A Sustainable Industrial Platform For Bio-Based Chemical Production

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Biotechnology Advances 31 (2013) 12001223

Contents lists available at ScienceDirect

Research review paper

Metabolic engineering of Escherichia coli: A sustainable industrial

Corresponding author. Tel./fax: +86 510 85918122.

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

Biopolymer and monomers production . . . . . . .

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

Titer and yield

Fuel and energy carrier

Fuel, solvent, food,

10-L bioreactor batch

Shake asks and IPTG

1.0 mol H2/mol

Bulk material, gasoline

30 g/L; 88% of the

9.5 g/L; 0.11 g/g

143 g/L; 67.4%

Gasoline blend stock,

1-L Bioreactor with

20 g/L; 86% of the

Deletion of negative regulator and

57.1 g/L; 0.83 g/g

99.2 g/L; 110%

90 g/L; 0.68 g/g

878 mM; 75% of

500-mL Flask with

Evaluation of various synthetic

Shake asks with IPTG

13.3 g/L; 0.1 g/g

5.6 g/L; 0.213 g/g

6.1 g/L; 0.31 g/g

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

Titer and yield

141.6 g/L; 4.63 g

Medicine, foods and

Constitutive expression of an operon

2700 g/g DCWd

Proudction rate of H2.

some E. coli metabolic regulators involved in resistance to furfural

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

minimal metabolic functionality was rationally designed and

ethanol, and tolerance to ethanol and other inhibitory compounds of

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

completely replace gasoline. Traditionally, 1-butanol was produced by

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

pathways, and the isoleucine biosynthetic pathway, respectively (Fig. 1)

2.3.3. Isopropanol and isobutanol production

Lactate (D- or L-), an important organic acid, is a bulk chemical

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

Zhou et al. (2012a) manipulated a wild-type E. coli B0013 by

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

manipulation compared to other standard strains such as E. coli K12 and

E. coli strain was developed that converted 40 g/L glycerol to 32 g/L

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

the enhanced production of succinic acid. Subsequently, inactivation

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

Therefore, metabolic engineering was further carried out to direct

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223

Pentose phosphate pahwtway

4.1. L-Threonine production

thrB gene is subject to feedback inhibition by L-threonine as homoserine

X. Chen et al. / Biotechnology Advances 31 (2013) 12001223