Available online at www.sciencedirect.
com
Advances in metabolic pathway and strain engineering paving the
way for sustainable production of chemical building blocks
Yun Chen1 and Jens Nielsen1,2,3
Bio-based production of chemical building blocks from
renewable resources is an attractive alternative to petroleum-
based platform chemicals. Metabolic pathway and strain
engineering is the key element in constructing robust microbial
chemical factories within the constraints of cost effective
production. Here we discuss how the development of
computational algorithms, novel modules and methods, omics-
based techniques combined with modeling refinement are
enabling reduction in development time and thus advance the
field of industrial biotechnology. We further discuss how recent
technological developments contribute to the development of
novel cell factories for the production of the building block
chemicals: adipic acid, succinic acid and 3-hydroxypropionic
acid.
Addresses
1
Novo Nordisk Foundation Center for Biosustainability, Department of
Chemical & Biological Engineering, Chalmers University of Technology,
SE412 96 Gothenburg, Sweden
2
Novo Nordisk Foundation Center for Biosustainability, Technical
University of Denmark, DK2970 Hørsholm, Denmark
3
Science for Life Laboratory, Royal Institute of Technology,
SE171 65 Solna, Sweden
Corresponding author: Nielsen, Jens (nielsenj@chalmers.se)
Current Opinion in Biotechnology 2013, 24:965–972
This review comes from a themed issue on Chemical biotechnology
Edited by Kristala LJ Prather
For a complete overview see the Issue and the Editorial
Available online 28th March 2013
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.copbio.2013.03.008
Introduction
Chemicals are an integral part of our daily life and play an
important role in the world economy. The global chemi-
cal industry has grown rapidly since 1970, and the revenue
it has generated increased about threefold over the last
decade, growing to USD5000B in 2011 (according to the
American Chemistry Council, URL: http://www.ameri-
canchemistry.com/Jobs/EconomicStatistics/Industry-Pro-
file/Global-Business-of-Chemistry). Although providing a
variety of useful products, the chemical industry is inher-
ently non-sustainable as it heavily relies on crude
petroleum and its environmentally damaging production
processes [1]. The desire to reduce dependency on
petroleum and an increased concern for the environment
are leading drivers for the sustainable production of
both fine and bulk chemicals from renewable resources.
www.sciencedirect.com
The high productivity of the current chemical industry
has contributed to efficient conversion of platform pet-
rochemicals into a broad array of industry chemical pro-
ducts. It is therefore of great interest to produce a number
of bio-based building block chemicals from biomass as a
renewable resource, to fit into the highly optimized
chemical manufacturing processes [2].
Microorganisms are increasingly used as platform cell
factories by industrial biotechnology to produce a whole
range of compounds that find numerous applications,
such as alcohols, organic acids, amino acids, pharmaceu-
ticals and polymers. However, the cost and required
platform techniques dictate that currently only a few
platform cell factories are preferred in industry, including
(but not limited to) the bacteria Escherichia coli, Coryne-
bacterium glutamicum and Bacillus subtilis, the filamentous
fungi Aspergillus niger, Aspergillus oryzae, Penicillium chry-
sogenum and the yeast Saccharomyces cerevisiae. Of these, S.
cerevisiae proves to be an attractive cell factory due to its
robustness and remarkable tolerance against various stres-
ses at industrial conditions. This cell factory has been
extensively used in food and beverage production, and is
also exploited for the production of fine and bulk chemi-
cals such as nutraceuticals and pharmaceuticals.
By far the most important reason for industry not to shift
from petroleum to bio-based production of chemicals is the
higher production cost. To cope with these constraints, an
integrated approach to constructing robust microbial
chemical factories and improved production processes
would be required (Figure 1). Available computational
algorithms allow systems level design [3]. Emerging tools
and methods in synthetic biology open up the possibility to
make metabolic pathway and strain engineering easier and
faster. Integrative analysis of comprehensive omics data
with modeling refinement facilitates multiple-objective
optimization. In this review, we will discuss how progress
in fundamental techniques advances metabolic pathway
and strain engineering, thereby affording the potential to
substantially reduce research and development time and to
decrease time to market. As examples of building block
chemicals, adipic acid, succinic acid and 3-hydroxypropio-
nic acid, which represent a near-term opportunity for the
replacement of petrochemicals with renewable resources,
will be discussed in detail.
Computational platforms guide systematic
design
The use of powerful computational tools can lead to
better-informed and more rapid design and optimization.
Current Opinion in Biotechnology 2013, 24:965–972
966 Chemical biotechnology

Figure 1

System design

Modules Methods
Fast assembly or engineering
RBS Plasmid Genome integration
TRMR
MAGE
SLIC

DATABASE Control of enzymes or pathways

Novel Enzyme

Post-transcription

n
Metagenomics

tio
ri p
(Re)Construction

sc
an
Tr
Protein Switch
Predicted

Evolution or
Library rational design
Measured

Omics-techniques Modeling refinement

Integrative

vb
analysis v
Fluxomics S = σi (UiVT)
Metabolomics i =0
va
Proteomics

vc
Transcriptomics v1 – v2 – v3 +1 –1 –1 0 0

Genomics – v1 + 2v2 – v4 = –1 +2 0 –1 0
v1 + v4 – v5 +1 0 0 +1 –1

MICROBIAL CHEMICAL FACTORY

Current Opinion in Biotechnology

Overview of integrated approach for construction of robust microbial chemical factories (MCF). Computational platforms guide systematic design.
Discovering and engineering novel modules and devices afford great potential for robust pathway construction. Emerging methods for fast assembly
and engineering, and tools for modulating gene expression and regulating metabolic pathway facilitate efficient construction. Systematic analysis of
comprehensive omics-data with modeling refinement enhances capability for global optimization. Iterative engineering cycles are needed to achieve
the desired MCF.

A range of computational pathway prediction algorithms
has been generated providing a systematic framework for
metabolic pathways (re)design, which is not only limited
to changing existing pathways through introduction of
gene knockouts or amplifications, such as OptKnock [4],
OptGene [5], OptForce [6] or FSEOF [7], but also aids in
identifying possible pathways from first principles based
on known enzyme reactions, for example, DESHARKY
[8] or based on possible biotransformations of functional
groups by known chemistry, for example, BNICE [9]. A
similarly principled predictor has identified more than
10 000 possible pathways for the synthesis of 1,4-butane-
diol from common central metabolites [10]. Therefore, it
is of primary importance not only to predict a wide range
of possible routes, but also to rank them based on dis-
criminative criteria. A prioritization scoring algorithm
based on binding covalence, chemical similarity, thermo-
dynamic favorability and pathway distance has been
suggested [11], and the recently published web server
RetroPath [12] offers a way to retrieve reactions varying in
number from the large numbers of reactions found using
BNICE to the small numbers of reactions that are pre-
sented in the KEGG database.
In addition, computer-aided design software for design-
ing regulatory circuits and aided protein engineering has

Current Opinion in Biotechnology 2013, 24:965–972 www.sciencedirect.com


been developed. An automated prediction algorithm can
predict the relative translation initiation rates of genes
with specific ribosome binding site (RBS) sequences, and
also enables design of synthetic RBS with a desired
translation rate [13]. The recently developed Rosetta
de novo design protocol is capable of generating enzyme
activity from an inert scaffold, rather than relying on an
existing catalytic activity as a starting point [14].
Novel modules and tools
Novel devices with high efficiency, such as novel
enzymes (genes), promoters and RNA-based control
modules, are prerequisites to construct and optimize
robust biological systems for chemical production. Avail-
ability and catalytic efficiency of enzymes are of critical
importance for metabolic pathway engineering. Through
mining the extensive genomic, metabolic and enzyme
databases such as KEGG, NCBI and SWISS-PROT to
identify required enzymes, for example, exploiting
natural substrate promiscuity led to identification of a
new amination enzyme for the synthesis of a non-natural
amino acid L-homoalanine [15] or subtractive genome
analysis was applied to identify the key enzymes for long
chain alkane production [16]. Besides, metagenome
screenings can extend the access to naturally existing
catalytic diversity [17]. Moreover, in addition to random
mutagenesis and recombination (shuffling), the develop-
ment of methods for evolving or rationally designing
enzymes will significantly expand the potential to create
novel enzymes with higher activity and specificity. Using
enzyme evolution and replacement of amino acids at the
active site of levopimaradiene synthase successfully
increased enzyme catalytic activity, resulting in 10-fold
increased levopimaradiene production [18].
Besides the identity of the enzymes (genes) selected,
novel modules are required for efficient construction and
fine-tuning expression. A number of constructs allow for
expression of target genes from plasmids, artificial or
native chromosomes. A variety of well-characterized pro-
moters exist for expression of target genes, and several
synthetic promoter libraries have been constructed by
mutagenesis of promoter regions through error-prone
PCR [19], or shuffling of multiple gene promoters [20].
This has significantly expanded the promoter selection
availability, but there is still a need for further develop-
ment in the field, for example, for inducible promoters for
yeast and for better characterization of known promoters
at different environmental conditions. A step in this
direction is RNA-based synthetic control modules allow-
ing for predictable tuning of expression levels [21].
Rapid engineering and qualitative control
The development of methods to rapidly assemble large
synthetic pathways provides a scalable alternative to
traditional recombinant DNA cloning methods. A notable
example is assembling and cloning a whole bacterial
www.sciencedirect.com
Sustainable production of chemicals Chen and Nielsen 967
genome in yeast by using a combination of enzymatic
in vitro assembly and in vivo homologous recombination
[22]. Ligation-free method SLIC and BglBricks have also
enabled rapid construction of operons and pathways
[23,24]. DNA assembler via in vivo homologous recom-
bination in yeast is capable of rapidly assembling multiple
genes in a single step [25]. Moreover, rapid engineering of
the chromosome of an organism was demonstrated using
multiplex automated genome engineering (MAGE) [26]
or trackable multiplex recombineering (TRMR) [27].
Using these methods and even more modules and devices
currently being developed, the time required for genetic
construction can be drastically reduced.
A diverse array of toolsets has been described for quali-
tatively tuning the expression of genes and regulating
pathway fluxes:
(i) Modifying gene dosage. A ubiquitin-based degra-
dation system was employed to modify the plasmid
copy number and gene dosage, resulting in threefold
improvement in patchoulol production [28].
(ii) Control of transcriptional activity. Modifying the
components of global transcription machinery
resulted in increased ethanol tolerance and pro-
duction in yeast [29].
(iii) Posttranscriptional processing. Rnt1p mediated hair-
pin was applied to titrate expression of the native
yeast ERG9 gene and control of flux through the
ergosterol pathway [21].
(iv) Spatial manipulation of enzymes. Direct enzyme
fusion [30], attachment to a protein or RNA-based
scaffolding system [31,32], or targeting to native
organelles [33] are gaining increasing attention, as
they can reduce the loss of intermediates via side
reactions, degradation or secretion, negative effects
of toxic intermediates, and unwanted allosteric
regulation.
(v) Switches that sense and respond to environmental
changes. DSRS is a recently developed system
which can dynamically detect specific metabolites in
the host and automatically regulate a metabolic
pathway. It was demonstrated that this DSRS
increased biodiesel production to 1.5 g L 1 and
improved the yield threefold [34].
These tools offer great potential for improving the con-
struction and control of biosynthetic pathways.
Integrative analysis contributes to global
analysis and optimization
Recent developments in comprehensive transcriptomics,
proteomics, posttranslational protein modifications and
metabolomics, combined with computational modeling
enable much detailed understanding of cellular and
metabolic characteristics. This knowledge, insight and
Current Opinion in Biotechnology 2013, 24:965–972
968 Chemical biotechnology
information will facilitate identification of target genes for
multiple-objective optimization. There are many success-
ful examples of metabolic pathways and strain engineering
using these analysis tools. With the help of genome scale
algorithms to identify gene knockout targets, cubebol
production was improved [35]. ChIP-chip was used to
identify novel candidate cofactor proteins interacting with
transcriptional regulator Tup1 helping to modulate the
cellular response to a variety of stress conditions [36].
Targeted proteomics was able to identify potential bottle-
necks in a mevalonate-based pathway for terpene pro-
duction [37]. 13C metabolic analysis was adopted to
identify bottleneck reactions for lysine production in C.
glutamicum [38]. DNA microarray and metabolite profiling
studies have revealed an imbalance in carbon flux due to
the accumulation of the toxic intermediate 3-hydroxy-3-
methyglutary-CoA in a synthetic pathway [39]. A multi-
variate-modular approach has been implemented to bal-
ance the metabolic fluxes for enhanced production of a
taxol precursor [40]. These achievements demonstrate that
the combination of systems level analysis with omics-based
techniques can be effective to overcome hidden problems
and optimize metabolic pathways and host organisms.
The data from systems-level analysis are captured and fed
back to refine modeling, and better-informed modeling
will in turn facilitate simulation and optimization of the
cell factory. As models expand and are used to integrate
omics data, the interpretation of the cellular character-
istics including the metabolism and regulatory network is
being continually upgraded. One example in this direc-
tion is the constraint-based reconstruction and analysis
(COBRA) approach, which has been recently reviewed
[41]. Computational tools are already guiding system
design and optimization with more and more successful
viable examples, and user-friendly simulation tools are
being developed, such as the BioMet Toolbox [42],
making this complex technique accessible to the non-
specialist experimental microbiologist. It is expected that
more accurate genome scale metabolic models will
increasingly be employed in metabolic engineering for
bio-based chemical production.
Combinational approaches for strain
improvement
To be competitive with petrochemical processes, it is
essential that microbial conversions are executed with
high productivity and yield, and that there is a significant
cost reduction of the raw material, for example, by using
inexpensive feedstocks, such as biomass. Much effort has
been exerted to extend the carbon substrate range and to
improve carbon utilization efficiency with a particular
focus on bio-based production of fuels and bulk chemi-
cals. To debottleneck yeast’s inability of digesting the
five-carbon sugars present in lignocellulosic biomass, in a
recent study combining metabolic with evolutionary
engineering new S. cerevisiae strains with greatly
Current Opinion in Biotechnology 2013, 24:965–972
improved anaerobic growth rate, xylose consumption rate
and ethanol production were generated [43]. In another
study the galactose uptake rate was increased by adaptive
evolution. In connection with the use of adaptive evol-
ution [44] subsequent systems level analysis is important,
as illustrated recently when the Ras2/PKA signaling path-
way was identified as a new target for improving galactose
utilization [45].
Another important factor that can impact the overall
process viability is product tolerance, as many chemicals
can adversely affect cell growth. Evolutionary engineer-
ing combined with functional genomics analysis of toler-
ance to exogenously applied products of interest has
shown that it is possible to discover potential resistance
mechanisms [46]. Another approach is to use computer
aided design of an efflux pump library followed by screen-
ing. This approach enabled discovery of a new efflux
pump that facilitated the tolerance to the target biofuel,
and enhanced limonene production twofold [47].
Production of valuable building block
chemicals
Currently available bio-based chemicals include com-
modity and fine chemicals, fuels and materials.The first
commercial examples, such as polylactic acid, polyhy-
droxyalkanoate and 1,3-propanediol, represent successful
market penetrations for large-scale bio-based products.
With the development in both the academic and indus-
trial fields, more and more bio-based chemicals are
becoming cost competitive representing a near-term
opportunity for the replacement of petrochemicals with
chemicals produced from renewable resources.
Adipic acid
Adipic acid is a building block chemical for nylon-6,6
polyamide and other polymers with an annual market over
USD6B. It is currently produced from benzene, a carcino-
genic petroleum fraction [48]. Verdezyne used synthetic
gene libraries to design a novel synthetic pathway in a yeast
to produce adipic acid from feedstocks such as sugars,
plant-based oils and paraffin [49]. The disruption of the
gene POX4 coding for acyl-CoA oxidase, which partially
blocks the beta-oxidation activity, and factors that redirect
the substrates to the terminal omega-oxidation were
employed to enhance production of this product [49]. This
process was demonstrated at a pilot plant scale from
vegetable oil (Verdezyne, URL: http://verdezyne.com/
verdezyne/News/documents/VerdezynePilotPlantRelea-
seBusinessFINAL.pdf).
Succinic acid
Succinic acid is a chemical intermediate widely used in
the detergent/surfactant and ion chelator market, as well
as an additive in the food and pharmaceutical market.
The current global market for succinic acid is about
30 000–50 000 tons per year with a market price of
www.sciencedirect.com

Figure 2
Glucose
Phosphoenolpyruvate
Anaplerotic pathway
Pyruvate
Acetyl-CoA
Oxaloacetate
Citrate
Malate
Glyoxylate pathway
Glyoxylate
Isocitrate
Fumarate
Succinate
Current Opinion in Biotechnology
Metabolic pathways for the production of succinic acid from glucose.
The anaplerotic pathway and glyoxylate pathway are presented in blue
and purple, respectively.
USD2400–3000 per ton, but the market potential is
expected to be more than 100 000 tons per year by
2015 [50]. To biotechnologically produce succinic acid
three metabolic pathways can be implemented: the
reductive branch of the TCA cycle, the oxidative branch
of the TCA cycle and the glyoxylate pathway (Figure 2).
There have been considerable research efforts predomi-
nantly using prokaryotic organisms, such as rumen bac-
teria and recombinant E. coli, to improve the bio-based
production of succinic acid [51,52]. One remarkable
example is the development of a recombinant E. coli
strain via rational design combined with adaptive evol-
ution, capable of producing 87 g L 1 succinic acid [52].
Succinate concentrations up to 146 g L 1 were achieved
in a cell recycling fed-batch culture of engineered C.
glutamicum [53].
Yeast and filamentous fungi have also been developed for
succinate production, since they can grow at rather low
pH which would make their cultivation more favorable
for downstream processing [54–56]. Raab et al. developed
a quadruple deletion S. cerevisiae strain, which could
produce succinic acid at a titer of 3.62 g L 1 with a yield
of 0.11 mol (mol glucose) 1 [55]. The yeast Yarrowia
lipolytica was engineered by blocking the succinate
dehydrogenase activity and combining with random
mutation for enhanced growth, capable of producing
succinate at the level of 45.5 g L 1 in buffered medium
www.sciencedirect.com
Sustainable production of chemicals Chen and Nielsen 969
Figure 3
DHAP DHA Glycerol
DhaB
G3P 3-HPA
AbSadH or
β-alanine puuC
Glucose
3-HP
α-alanine
CaMCR
Pyruvate Malonate
semialdehyde
Lactate
CaMCR
Acetyl-CoA Malonyl-CoA
Lactoyl-CoA Acryloyl-CoA 3-HP-CoA
Current Opinion in Biotechnology
Metabolic pathway through beta-alanine (red), glycerol (blue), malonyl-
CoA (green) or lactate (purple) to produce 3-hydroxypropionic acid.
and 17.4 g L 1 with the final pH dropping to a value of
about 3 [56]. Reverdia, a company backed by DSM and
Roquette, has developed a low-pH (pH = 3) yeast-based
fermentation process to produce biosucciniumTM from
renewable carbon sources (glucose and carbon dioxide),
which has been successfully produced in a 300 metric
tonne capacity demonstration plant [57]. For microbial
production of building block chemicals anaerobic fermen-
tation processes have attracted interest since they nor-
mally result in higher theoretical yields [58,59]. An
engineered yeast expressing heterologous phosphoenol-
pyruvate carboxykinase, malate dehydrogenase and
fumarase in the cytosol was used in an anaerobic process
at low-pH conditions for the production of dicarboxylic
acids such as succinic acid with ethanol as a minor by-
product [59].
3-Hydroxypropionic acid
3-Hydroxypropionic acid (3-HP) is a platform chemical
that has recently received significant attention due to its
numerous applications. In the chemistry industry it can
be used to produce a range of valuable chemicals and
polymers, of which acrylic acid is the most attractive
compound. Other derivatives include acrylamide, acrylo-
nitrile, 1,3-propanediol, and malonic acid. Since no organ-
ism has been identified so far that naturally produces 3-
HP as a major product, a number of biosynthetic pathways
have been suggested for the production of 3-HP from
renewable resources [60]. Henry and coworkers demon-
strated with the help of BNICE that it was possible to
design novel biosynthetic pathways for the production of
3-HP from pyruvate [61]. One of these pathways through
beta alanine (Figure 3) has been implemented in an
industrial setting, after obtaining the required novel
aminotransferase through protein evolution engineering
[62,63].
Current Opinion in Biotechnology 2013, 24:965–972
970 Chemical biotechnology
Significant efforts have been made to develop a recom-
binant E. coli strain for the production of 3-HP from
glycerol (Figure 3). In order to balance the enzyme
activity and stabilize glycerol dehydratase (DhaB), heter-
ologous DhaB and DhaB reactivase from Klebsiella pneu-
monia and an alpha-ketoglutaric semialdehyde
dehydrogenase (AbSadH) from Azospirillum brasilense
were employed to reconstruct the 3-HP biosynthetic
pathway in E. coli. Consequently, the highest described
titer was obtained at 38.7 g L 1, with an average carbon
yield of 35% on glycerol under aerobic conditions [64].
This process, however, requires the addition of vitamin
B12 to the culture medium, making it unfeasible for
industrial applications. Current efforts are geared toward
using K. pneumonia as an alternative host, since it can
produce vitamin B12 naturally [65,66]. In recent studies
by redirecting the metabolic flux toward 3-HP production
with proper fermentation condition optimization, a 3-HP
titer of more than 28 g L 1 was achieved [67,68].
Considering the utilization of lignocellulosic sugars, one
pathway, namely the malonyl-CoA pathway (Figure 3),
was investigated in both academia and industry [69,70].
The biosynthetic pathway was primarily reconstructed in
E. coli leading to reduction of the intermediate metabolite
malonyl-CoA. By overexpressing endogenous acetyl-CoA
carboxylase (ACC) and nicotinamide nucleotide transhy-
drogenase (PntAB), an engineered E. coli expressing
malonyl-CoA reductase (CaMCR) from Chloroflexus aur-
antiacus was able to produce 3-HP at the level of
193 mg L 1 [69]. With the help of methodologies, such
as SCALEs [71], improved 3-HP tolerance was obtained
by overexpression of a few genes and by deletion of a
number of 3-HP tolerance complex repressor genes (tyrR,
trpR, metJ, purr, lysR, nrdR and equivalents) [70,72].
Compared to E. coli, yeast is a suitable cell factory for the
production of organic acids such as 3-HP since it tolerates
low pH, which greatly reduces impurities and undesired
by-products. So far only one pathway, through lactate, has
been examined in yeast composed of propionyl-CoA
transferase, lactyl-CoA dehydratase, 3-hydroxypropio-
nyl-CoA dehydratase and 3-hydroxypropionyl-CoA
hydrolase to produce 3-HP (Figure 3) [73].
Perspective
Although there are many commercially successful bio-
based chemical products, and more and more biochemical
products continue to penetrate diverse industrial markets,
bio-based chemical production is still in its commercial
infancy. A number of hurdles to realize the commercia-
lization of biochemical production still remain, with the
key factors to market success being price competitiveness
and environmental sustainability. Reducing the costs of
biochemical production depends on low costs of the raw
material and also depends strongly on reducing the costs
of the process. In this sense, the long-term growth of
Current Opinion in Biotechnology 2013, 24:965–972
bio-based chemical products will depend on the devel-
opment of cost-competitive bioprocess in terms of titers,
yields and productivities. Advances in synthetic biology,
systems biology and evolutionary engineering, and our
continually improving understanding of metabolism and
developing tools for its manipulation, can be expected to
help to bring down the overall bioprocess cost and
improve the chance of success. Besides there are increas-
ing industry and market pulls for bio-based chemical
production. We believe that further developments in
metabolic pathway and strain engineering will finally
contribute to the creation of a more sustainable and
environmentally friendly chemical industry.
Acknowledgments
We would like to thank Dr Verena Siewers, Yongjin Zhou, Luis Caspeta-
Guadarrama, Rahul Kumar for reading the manuscript and providing many
valuable comments. This work has been funded by Vinnova, the Chalmers
Foundation, the Knut and Alice Wallenberg Foundation, European
Research Council project INSYSBIO (Grant no. 247013) and the Novo
Nordisk Foundation.
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Dhamankar H, Prather KL: Microbial chemical factories: recent
advances in pathway engineering for synthesis of value added
chemicals. Curr Opin Struct Biol 2011, 21:488-494.
2. Nikolau BJ, Perera MA, Brachova L, Shanks B: Platform
biochemicals for a biorenewable chemical industry. Plant J
2008, 54:536-545.
3. Kim IK, Roldão A, Siewers V, Nielsen J: A systems-level
approach for metabolic engineering of yeast cell factories.
FEMS Yeast Res 2012, 12:228-248.
4. Patil KR, Rocha I, Forster J, Nielsen J: Evolutionary
programming as a platform for in silico metabolic engineering.
BMC Bioinformatics 2005, 6:308.
5. Pharkya P, Burgard AP, Maranas CD: OptStrain: a computational
framework for redesign of microbial production systems.
Genome Res 2004, 14:2367-2376.
6. Ranganathan S, Suthers PF, Maranas CD: OptForce: an
optimization procedure for identifying all genetic
manipulations leading to targeted overproductions. PLoS
Comput Biol 2010, 6:e1000744.
7. Choi HS, Lee SY, Kim TY, Woo HM: In silico identification of
gene amplification targets for improvement of lycopene
production. Appl Environ Microbiol 2010, 76:3097-3105.
8. Rodrigo G, Carrera J, Prather KJ, Jaramillo A: DESHARKY:
automatic design of metabolic pathways for optimal cell
growth. Bioinformatics 2008, 24:2554-2556.
9. Hatzimanikatis V, Li C, Ionita JA, Henry CS, Jankowski MD,
Broadbelt L: Exploring the diversity of complex metabolic
networks. Bioinformatics 2005, 21:1603-1609.
10. Yim H, Haselbeck R, Niu W, Pujol-Baxley C, Burgard A, Boldt J,
Khandurina J, Trawick JD, Osterhout RE, Stephen R et al.:
Metabolic engineering of Escherichia coli for direct
production of 1,4-butanediol. Nat Chem Biol 2011, 7:445-452.
11. Cho A, Yun H, Park JH, Lee SY, Park S: Prediction of novel
 synthetic pathways for the production of desired chemicals.
BMC Syst Biol 2010, 4:35.
This paper reports development of prioritization scoring algorithms for
identifying novel pathways for the production of chemicals of interest.
www.sciencedirect.com

12. Carbonell P, Planson AG, Fichera D, Faulon JL: A retrosynthetic
biology approach to metabolic pathway design for therapeutic
production. BMC Syst Biol 2011, 5:122.
13. Salis HM, Mirsky EA, Voigt CA: Automated design of synthetic
ribosome binding sites to control protein expression. Nat
Biotechnol 2009, 27:946-950.
14. Richter F, Leaver-Fay A, Khare SD, Bjelic S, Baker D: De novo
enzyme design using Rosetta3. PLoS ONE 2011, 6:e19230.
15. Zhang K, Li H, Cho KM, Liao JC: Expanding metabolism for total
biosynthesis of the nonnatural amino acid L-homoalanine.
Proc Natl Acad Sci U S A 2010, 107:6234-6239.
16. Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB: Microbial
 biosynthesis of alkanes. Science 2010, 329:559-562.
An impressive illustration of how mining the extensive databases can be
used to identify the key enzymes for the production of target product.
17. Hess M, Sczyrba A, Egan R, Kim TW, Chokhawala H, Schroth G,
Luo S, Clark DS, Chen F, Zhang T et al.: Metagenomic discovery
of biomass-degrading genes and genomes from cow rumen.
Science 2011, 331:463-467.
18. Leonard E, Ajikumar PK, Thayer K, Xiao WH, Mo JD, Tidor B,
Stephanopoulos G, Prather KLJ: Combining metabolic and
protein engineering of a terpenoid biosynthetic pathway for
overproduction and selectivity control. Proc Natl Acad Sci U S A
2010, 107:13654-13659.
19. Alper H, Fischer C, Nevoigt E, Stephanopoulos G: Tuning genetic
control through promoter engineering. Proc Natl Acad Sci U S A
2005, 102:12678-12683.
20. Lu C, Jeffries T: Shuffling of promoters for multiple genes to
optimize xylose fermentation in an engineered
Saccharomyces cerevisiae strain. Appl Environ Microbiol 2007,
73:6072-6077.
21. Babiskin AH, Smolke CD: A synthetic library of RNA control
 modules for predictable tuning of gene expression in yeast.
Mol Syst Biol 2011, 7:471.
An example of RNA-based control elements allow for predictable tuning
of expression levels, demonstrated with systematic titration of flux
through the ergostrol pathway in yeast.
22. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY,
Algire MA, Benders GA, Montague MG, Ma L, Moodie MM et al.:
Creation of a bacterial cell controlled by a chemically
synthesized genome. Science 2010, 329:52-56.
23. Li MZ, Elledge SJ: Harnessing homologous recombination in
vitro to generate recombinant DNA via SLIC. Nat Methods 2007,
4:251-256.
24. Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin AP,
Keasling JD: BglBricks: a flexible standard for biological part
assembly. J Biol Eng 2010, 4:1.
25. Shao Z, Luo Y, Zhao H: Rapid characterization and engineering
of natural product biosynthetic pathways via DNA assembler.
Mol Biosyst 2011, 7:1056-1059.
26. Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR,
Church GM: Programming cells by multiplex genome
engineering and accelerated evolution. Nature 2009,
460:894-898.
27. Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LB, Gill RT:
 Rapid profiling of a microbial genome using mixtures of
barcoded oligonucleotides. Nat Biotechnol 2010, 28:856-862.
This strategy enables simultaneously engineering and monitoring thou-
sands of genes in a target organism.
28. Chen Y, Partow S, Scalcinati G, Siewers V, Nielsen J: Enhancing
the copy number of episomal plasmids in Saccharomyces
cerevisiae for improved protein production. FEMS Yeast Res
2012, 12:598-607.
29. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G:
Engineering yeast transcription machinery for improved
ethanol tolerance and production. Science 2006,
314:1565-1568.
30. Albertsen L, Chen Y, Bach LS, Rattleff S, Maury J, Brix S, Nielsen J,
Mortensen UH: Diversion of flux toward sesquiterpene
www.sciencedirect.com
Sustainable production of chemicals Chen and Nielsen 971
production in Saccharomyces cerevisiae by fusion of host and
heterologous enzymes. Appl Environ Microbiol 2011,
77:1033-1040.
31. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ,
Ullal AV, Prather KL, Keasling JD: Synthetic protein scaffolds
provide modular control over metabolic flux. Nat Biotechnol
2009, 27:753-759.
32. Delebecque CJ, Lindner AB, Silver PA, Aldaye FA: Organization
 of intracellular reactions with rationally designed RNA
assemblies. Science 2011, 333:470-474.
A RNA-based scaffolding system was demonstrated to control the spatial
organization of a hydrogen-producing pathway, resulting in 48-fold
increase in hydrogen production.
33. Farhi M, Marhevka E, Masci T, Marcos E, Eyal Y, Ovadis M,
Abeliovich H, Vainstein A: Harnessing yeast subcellular
compartments for the production of plant terpenoids. Metab
Eng 2011, 13:474-481.
34. Zhang F, Carothers JM, Keasling JD: Design of a dynamic
 sensor-regulator system for production of chemicals and
fuels derived from fatty acids. Nat Biotechnol 2012, 30:354-359.
Presentation of an excellent strategy that uses biosensors dynamically
adjusting the metabolic pathway according to the metabolic status of the
host.
35. Asadollahi MA, Maury J, Patil KR, Schalk M, Clark A, Nielsen J:
Enhancing sesquiterpene production in Saccharomyces
cerevisiae through in silico driven metabolic engineering.
Metab Eng 2009, 11:328-334.
36. Hanlon SE, Rizzo JM, Tatomer DC, Lieb JD, Buck MJ: The stress
response factors Yap6, Cin5, Phd1, and Skn7 direct targeting
of the conserved co-repressor Tup1-Ssn6 in S. cerevisiae.
PLoS ONE 2011, 6:e19060.
37. Redding-Johanson AM, Batth TS, Chan R, Krupa R, Szmidt HL,
 Adams PD, Keasling JD, Soon Lee T, Mukhopadhyay A,
Petzold CJ: Targeted proteomics for metabolic pathway
optimization: application to terpene production. Metab Eng
2011, 13:194-203.
Demonstration of how proteomics is advancing towards discovering
potential bottlenecks and optimizing metabolic pathway.
38. Becker J, Zelder O, Hafner S, Schroder H, Wittmann C: From zero
to hero-design-based systems metabolic engineering of
Corynebacterium glutamicum for L-lysine production. Metab
Eng 2011, 13:159-168.
39. Kizer L, Pitera DJ, Pfleger BF, Keasling JD: Application of
functional genomics to pathway optimization for increased
isoprenoid production. Appl Environ Microbiol 2008,
74:3229-3241.
40. Ajikumar PK, Xiao WH, Tyo KEJ, Wang Y, Simeon F, Leonard E,
Mucha O, Phon TH, Pfeifer B, Stephanopoulos G: Isoprenoid
pathway optimization for Taxol precursor overproduction in
Escherichia coli. Science 2010, 330:70-74.
41. Lewis NE, Nagarajan H, Palsson BO: Constraining the metabolic
genotype–phenotype relationship using a phylogeny of in
silico methods. Nat Rev Microbiol 2012, 10:291-305.
42. Cvijovic M, Olivares-Hernandez R, Agren R, Dahr N,
Vongsangnak W, Nookaew I, Patil KR, Nielsen J: BioMet toolbox:
genome-wide analysis of metabolism. Nucleic Acids Res 2010,
38:W144-W149.
43. Zhou H, Cheng J-s, Wang BL, Fink GR, Stephanopoulos G: Xylose
 isomerase overexpression along with engineering of the
pentose phosphate pathway and evolutionary engineering
enable rapid xylose utilization and ethanol production by
Saccharomyces cerevisiae. Metab Eng 2012, 14:611-622.
Illustration of how rational design combined with evolutionary engineering
is facilitating strain development with application to increase xylose
consumption rate and ethanol production.
44. Çakar ZP, Turanli-Yildiz B, Alkim C, Yilmaz U: Evolutionary
engineering of Saccharomyces cerevisiae for improved
industrially important properties. FEMS Yeast Res 2012,
12:171-182.
45. Hong KK, Vongsangnak W, Vemuri GN, Nielsen J: Unravelling
 evolutionary strategies of yeast for improving galactose
Current Opinion in Biotechnology 2013, 24:965–972
972 Chemical biotechnology
utilization through integrated systems level analysis. Proc Natl
Acad Sci U S A 2011, 108:12179-12184.
This study highlights the importance of integrative analysis with omics-
techniques as a key part in linking phenotype and genotype, and identify-
ing novel targets for strain improvement.
46. Atsumi S, Wu TY, Machado IM, Huang WC, Chen PY, Pellegrini M,
Liao JC: Evolution, genomic analysis, and reconstruction of
isobutanol tolerance in Escherichia coli. Mol Syst Biol 2010,
6:449.
47. Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD,
Hadi MZ, Mukhopadhyay A: Engineering microbial biofuel
tolerance and export using efflux pumps. Mol Syst Biol 2011,
7:487.
48. Polen T, Spelberg M, Bott M: Toward biotechnological
production of adipic acid and precursors from biorenewables.
J Biotechnol 2012 http://dx.doi.org/10.1016/
j.jbiotec.2012.1007.1008.
49. Picataggio S, Beardslee T. Biological methods for preparing adipic
acid. US patent 2012, US 8,241,879 B2.
50. Cheng KK, Zhao XB, Zeng J, Zhang JA: Biotechnological
production of succinic acid: current state and perspectives.
Biofuel Bioprod Bior 2012, 6:302-318.
51. Meynial-Salles I, Dorotyn S, Soucaille P: A new process for the
continuous production of succinic acid from glucose at high
yield, titer, and productivity. Biotechnol Bioeng 2008, 99:129-
135.
52. Jantama K, Haupt MJ, Svoronos SA, Zhang X, Moore JC,
Shanmugam KT, Ingram LO: Combining metabolic engineering
and metabolic evolution to develop nonrecombinant strains of
Escherichia coli C that produce succinate and malate.
Biotechnol Bioeng 2008, 99:1140-1153.
53. Okino S, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H: An
efficient succinic acid production process in a metabolically
engineered Corynebacterium glutamicum strain. Appl
Microbiol Biotechnol 2008, 81:459-464.
54. Gallmetzer M, Meraner J, Burgstaller W: Succinate synthesis and
excretion by Penicillium simplicissimum under aerobic and
anaerobic conditions. FEMS Microbiol Lett 2002, 210:221-225.
55. Raab AM, Gebhardt G, Bolotina N, Weuster-Botz D, Lang C:
Metabolic engineering of Saccharomyces cerevisiae for the
biotechnological production of succinic acid. Metab Eng 2010,
12:518-525.
56. Yuzbashev TV, Yuzbasheva EY, Sobolevskaya TI, Laptev IA,
Vybornaya TV, Larina AS, Matsui K, Fukui K, Sineoky SP:
Production of succinic acid at low pH by a recombinant strain
of the aerobic yeast Yarrowia lipolytica. Biotechnol Bioeng
2010, 107:673-682.
57. Smidt M: A sustainable supply of succinic acid.
EuroBiotechNews 2011, 10:11-12.
58. Litsanov B, Brocker M, Bott M: Toward homosuccinate
fermentation: metabolic engineering of Corynebacterium
glutamicum for anaerobic production of succinate from
glucose and formate. Appl Environ Microbiol 2012,
78:3325-3337.
Current Opinion in Biotechnology 2013, 24:965–972
59. Jansen M, Graaf M, Verwaal R. Dicarboxylic acid production
process. US patent 2012, US 2012/0040422 A1.
60. Jiang X, Meng X, Xian M: Biosynthetic pathways for 3-
hydroxypropionic acid production. Appl Microbiol Biotechnol
2009, 82:995-1003.
61. Henry CS, Broadbelt LJ, Hatzimanikatis V: Discovery and
analysis of novel metabolic pathways for the biosynthesis of
industrial chemicals: 3-hydroxypropanoate. Biotechnol Bioeng
2010, 106:462-473.
62. Liao H, Gokarn R, Gort S, Jessen H, Selifonova O. Alanine 2,3-
aminomutase. US patent 2007, US 7,309,597B2.
63. Liao H, Gokarn R, Gort S, Jessen H, Selifonova O. Production of 3-
hydroxypropionic acid using beta-alanine/pyruvate
aminotransferase. US patent 2010, US 2010/0136638 A1.
64. Rathnasingh C, Raj SM, Jo JE, Park S: Development and
evaluation of efficient recombinant Escherichia coli strains for
the production of 3-hydroxypropionic acid from glycerol.
Biotechnol Bioeng 2009, 104:729-739.
65. Ashok S, Raj SM, Rathnasingh C, Park S: Development of
recombinant Klebsiella pneumoniae DdhaT strain for the co-
production of 3-hydroxypropionic acid and 1,3-propanediol
from glycerol. Appl Microbiol Biotechnol 2011, 90:1253-1265.
66. Huang Y, Li Z, Shimizu K, Ye Q: Simultaneous production of 3-
hydroxypropionic acid and 1,3-propanediol from glycerol by a
recombinant strain of Klebsiella pneumoniae. Bioresour
Technol 2012, 103:351-359.
67. Ashok S, Sankaranarayanan M, Ko Y, Jae KE, Ainala SK, Kumar V,
Park S: Production of 3-hydroxypropionic acid from glycerol
by recombinant Klebsiella pneumoniae DdhaTDyqhD which
can produce vitamin B(12) naturally. Biotechnol Bioeng 2013,
110:511-524.
68. Ashok S, Mohan Raj S, Ko Y, Sankaranarayanan M, Zhou S,
Kumar V, Park S: Effect of puuC overexpression and nitrate
addition on glycerol metabolism and anaerobic 3-
hydroxypropionic acid production in recombinant Klebsiella
pneumoniae DglpKDdhaT. Metab Eng 2013, 15:10-24.
69. Rathnasingh C, Raj SM, Lee Y, Catherine C, Ashok S, Park S:
Production of 3-hydroxypropionic acid via malonyl-CoA
pathway using recombinant Escherichia coli strains. J
Biotechnol 2012, 157:633-640.
70. Lynch M, Gill RT, Warnecke-Lipscomb T. Method for producing 3-
hydroxypropionic acid and other products. PCT patent 2011, WO
2011/038364 A1.
71. Lynch MD, Warnecke T, Gill RT: SCALEs: multiscale analysis of
library enrichment. Nat Methods 2007, 4:87-93.
72. Warnecke-Lipscomb TE, Lynch M, Gill RT. Methods, systems and
compositions for increased microorganism tolerance to and
production of 3-hydroxypropionic acid (3-HP). PCT patent 2010,
WO 2010/011874 A2.
73. Gokarn R, Selifonova OV, Gort S, Selmer T, Buckel W. 3-
Hydroxypropionic acid an other organic compounds. US patent
2011, US 8,076,120B2.
www.sciencedirect.com