Biotechnology Advances 31 (2013) 976–985

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Combinatorial genetic perturbation to refine metabolic circuits

for producing biofuels and biochemicals
Hyo Jin Kim, Timothy Lee Turner, Yong-Su Jin ⁎
Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Department of Food Science and Human Nutrition, University of Illinois at Urbana–Champaign, 1206 West Gregory Dr., Urbana, IL 61801, USA

a r t i c l e i n f o a b s t r a c t

Available online 5 April 2013 Recent advances in metabolic engineering have enabled microbial factories to compete with conventional
processes for producing fuels and chemicals. Both rational and combinatorial approaches coupled with syn-
Keywords: thetic and systematic tools play central roles in metabolic engineering to create and improve a selected mi-
Combinatorial library crobial phenotype. Compared to knowledge-based rational approaches, combinatorial approaches exploiting
Inverse metabolic engineering biological diversity and high-throughput screening have been demonstrated as more effective tools for im-
Metabolic circuit
proving various phenotypes of interest. In particular, identification of unprecedented targets to rewire met-
Biofuel
Biochemical
abolic circuits for maximizing yield and productivity of a target chemical has been made possible. This review
highlights general principles and the features of the combinatorial approaches using various libraries to im-
plement desired phenotypes for strain improvement. In addition, recent applications that harnessed the com-
binatorial approaches to produce biofuels and biochemicals will be discussed.
© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 976
2. Major strategies for metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 977
2.1. Rational approaches for engineering phenotypes of target microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 977
2.2. Combinatorial approaches for engineering phenotypes of target microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . 978
3. Single gene overexpression and deletion libraries: mines of new discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 979
3.1. Examples of combinatorial genetic perturbations for producing fuels and chemicals . . . . . . . . . . . . . . . . . . . . . . . . . 980
3.2. Considerations for constructing genetic perturbation libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 980
4. Coordination of single or multi gene(s) expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981
4.1. Modulation of transcription via promoter modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981
4.2. Modulation of transcription via engineering RNA-based control modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981
5. Orchestration of global transcriptome through simultaneous perturbation of multiple gene targets . . . . . . . . . . . . . . . . . . . . . 982
5.1. Global transcription machinery engineering (gTME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982
5.2. Artificial transcription factor (ATF) libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982
5.3. Multiplexed automated genome engineering (MAGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
6. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983

1. Introduction resources for manufacturing biofuels and commodity biochemicals

Microbial factories implemented through metabolic engineering ap-
proaches based on systems and synthetic biology tools are astonishing
⁎ Corresponding author at: Institute for Genomic Biology, University of Illinois at Urbana–
Champaign, Urbana, IL 61801, USA. Tel.: +1 217 333 7981; fax: +1 217 333 0508.
E-mail address: ysjin@illinois.edu (Y.-S. Jin).
from renewable biomass (Clomburg and Gonzalez, 2010; Colin et al.,
2011; Gowen and Fong, 2011; Hong and Nielsen, 2012; Li et al., 2010;
Liu, 2011; Mukhopadhyay et al., 2008; Wackett, 2008). These microbial
factories have been preferred because they can produce target
molecules stereo-specifically with minimal amounts of unwanted
byproducts and offer easier purification processes as compared to con-
ventional chemical synthesis (Blixt and Razi, 2006; Takayama et al.,

0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.03.010
 H.J. Kim et al. / Biotechnology Advances 31 (2013) 976–985
1997). Additionally, operation of microbial factories could be safer than
that of chemical processes that often produce potentially toxic interme-
diates or byproducts. These are attractive characteristics of microbial
factories. Therefore, biochemicals produced through microbial factories
are likely more amenable for high-value products, such as
nutraceuticals or therapeutics. Most importantly, biological processes
based on microbial factories are more environmentally friendly because
these microbe-based processes not only emit less pollutants than tradi-
tional chemical processes, but also, net-production of CO2 of the biolog-
ical processes would be near zero as they use substrate produced via
photosynthesis. It is also anticipated that microbial factories using
plant biomass as feedstock will replace petroleum-based chemical
production processes due to uncertainty in supply and continuously
increasing costs of fossil fuels (Alper and Stephanopoulos, 2009;
Schmer et al., 2008; Sims et al., 2010; Sticklen, 2008; Wackett, 2008;
Zinoviev et al., 2010). Lastly, microbial factories are capable of producing
complex biochemicals that cannot be economically synthesized by
chemical processes (Ajikumar et al., 2010; Ro et al., 2006). Owed to
these numerous advantages, the development of design principles and
application tools for establishing microbial factories has drawn signifi-
cant amounts of attention during the last decade. Successful implemen-
tation of microbial factories for producing biofuels and biochemicals will
alleviate growing energy issues and improve human welfare.
Selection of a proper microbial host is the first and most critical step
of metabolic engineering to produce target biofuels and biochemicals
(Fig. 1). Various aspects of host phenotypes need to be considered: sub-
strate utilization capability, tolerance to products, optimum tempera-
ture and pH, etc. (Alper and Stephanopoulos, 2009). Once the host
microorganism is selected, genetic circuits in the host must be rewired
through overexpression or deletion of endogenous genes to establish
target phenotypes. A model microorganism, such as Escherichia coli or
Chassis Approach
- Eukaryote or - Rational metabolic
prokaryote engineering
- Imported or Innate - Evolutionary strategy
- Inverse metabolic
Resource
engineering
- Sugars
- Starches, lignocellulosics or other feedstocks
Fig. 1. Conceptual diagram representing microbial factory improvement steps for manufacturing biofuels and biochemicals. Selection of the feedstock is followed by choosing a suitable
microbial host. The strain can be optimized for production though various metabolic engineering approaches coupled with analytical tools.
977
Saccharomyces cerevisiae, is preferentially selected as the host due to
the availability of convenient and numerous genetic engineering tools.
In this scenario, introduction of heterologous pathways and control of
energy and redox balances are necessary to deliver optimal strains capa-
ble of producing biofuels and biochemicals efficiently and rapidly. Com-
bined with systems biology and bioinformatics, analytical tools play
pivotal roles to understand complex metabolic networks related to the
production of target molecules. Iterative and cooperative combination
between the analytical tools and strain engineering strategies is of impor-
tance for successful metabolic engineering (Blazeck and Alper, 2010).
Metabolic engineering strategies to improve the production of
biofuels and biochemicals by engineered strains are largely divided
into a rational and a combinatorial approach. In the beginning, this
review compares rational and combinatorial approaches. The combi-
natorial approach can be divided again into an evolutionary strategy
where beneficial spontaneous or induced mutations in the genome are
perused and a combinatorial library approach where genetic libraries
are selected or screened. In particular, this review focuses exclusively
on three approaches using combinatorial libraries for improving pheno-
types of interest. General principles and characteristics of each combina-
torial library approach as well as outcomes from recent applications for
improvement of microbial factories for producing biofuels and biochem-
icals are discussed (Table 1).
2. Major strategies for metabolic engineering
2.1. Rational approaches for engineering phenotypes of
target microorganisms
The most widely used metabolic engineering strategy is a rational
approach. The rational approach coordinates metabolic fluxes through
Biofuels and biochemicals
- Ethanol
- n-butanol, 2-butanol,
terpenoids or higher lipids
Analysis
- Genome sequencing
- Transcriptomics, proteomics
and metabolomics
- Bioinformatic tools
978 H.J. Kim et al. / Biotechnology Advances 31 (2013) 976–985

Table 1
Examples of inverse metabolic engineering to improve microbes for producing biofuels and biochemicals.

Product Resource Chassisa Strategyb Result Reference

Ethanol Galactose SC GPL A 250% increase in both galactose consumption rate and ethanol productivity Lee et al. (2011)
Ethanol Xylose SC GPL Ethanol yields improved from 0.1 to 0.3 g ethanol/g xylose Kim et al. (2012b)
Lycopene Glucose EC GPL Accumulation of 16,000 ppm (16 mg lycopene/g cell) of lycopene within 24 h Jin and Stephanopoulos (2007)
with 5 g/L of glucose
Ethanol Xylose SC GPL A 100% increase in the growth rate and a 70% increase in ethanol production Jin et al. (2005)
Ethanol, Isobutanol Glucose SC GPL A 3-fold increase in the specific growth rate under high levels of glucose (10%) Hong et al. (2010)
and ethanol (5%)
Ethanol Glucose SC GPL Normal growth under 41 °C Shahsavarani et al. (2012)
n-Butanol LB EC GPL Increased n-butanol tolerance by as much as 50% Reyes et al. (2011)
N/A LB EC GPL 130% increase in specific growth at inhibitory acetate concentration Sandoval et al. (2011)
Butanol Glucose CA GPL 81% increase in butanol tolerance Borden and Papoutsakis (2007)
N/A N/A CA GPL About 10-fold increase of growth in 1.4% (v/v) butyrate stress Borden et al. (2010)
Lycopene Glucose EC KOL 8.5-Fold increase of lycopene production Alper et al. (2005c)
Lycopene Glucose EC KOL A 40% increase in lycopene production (Accumulation of 6600 ppm Alper et al. (2005a)
(6.6 mg lycopene/g cell) of lycopene)
Lycopene Glucose EC PL Accumulation of approximately 2500 ppm (2.5 mg lycopene/g cell) of lycopene Alper et al. (2005a)
Glycerol Glucose SC PL A 40% increase in glycerol yield Nevoigt et al. (2006)
Mevalonate Glycerol EC TIGR A 7-fold increase in mevalonate production Pfleger et al. (2006)
n-Hexanol Glucose EC TEML 67% increase in n-hexanol production Machado et al. (2012)
N/A Xylose SC TEML 70% improvement of growth rate on xylose Young et al. (2012)
Isobutanol Glucose EC TEML A 3-fold increase in isobutanol yield and a 88% increase in specific productivity Bastian et al. (2011)
(g/L h−1 OD−1)
Ethanol Glucose SC gTME A 69% increase of volumetric ethanol productivity (g/L h−1) Alper et al. (2006)
Lycopene Glucose EC gTME Volumetric production (mg/L) improved from 4200 ppm (4.2 mg lycopene/g cell) Alper and Stephanopoulos (2007)
to 7700 ppm (7.7 mg lycopene/g cell)
Lycopene N/A EC MAGE A 5-fold increase in lycopene production (~9000 ppm, 9 mg lycopene/g cell) Wang et al. (2009)
N/A Corn stover EC TRMR 250% improved growth on hydrolysate corn stover Warner et al. (2010)
a
Abbreviated names of organisms: SC, S. cerevisiae; EC, E. coli; CA, Clostridium acetobutylicum.
b
Abbreviated names of inverse metabolic engineering strategies: TIGR, tunable intergenic regions library; GPL, genome-wide perturbation library; TEML, transporter (enzyme)
mutation library; gTME, global transcription machinery engineering library; KOL, knockout library; PL, promoter library; MAGE, multiplexed automated genome engineering;
TRMR, trackable multiplex recombineering.

intended manipulation of selected gene targets (transporters, enzymes
and/or regulatory proteins) based on prior knowledge (Stephanopoulos,
1999). Deduction of a possible flux-limiting step from available informa-
tion, and genetic perturbation of a gene(s) responsible for the identified
flux-limiting step are the two most important processes of the rational
approach. Although the rational approach is a feasible methodology to
generate engineered strains capable of producing target chemicals at
sub-optimal levels, this approach often allows limited success for com-
mercial applications (Bailey et al., 2002). This may be because the bio-
chemical reactions in a microbe are not governed by a single gene but
rather orchestrated by many genes or even intricate interactions among
them. Additionally, limited success using a rational approach may be par-
tially due to incomplete understanding of interactions of critical circuits
among the biomolecular network.
2.2. Combinatorial approaches for engineering phenotypes of
target microorganisms
Engineered strains exhibiting sub-optimal phenotypes using rational
approaches can be further improved through evolutionary strategies.
Selection or screening of mutant libraries containing spontaneous or
induced mutation is a classic method to achieve desired phenotypes.
Genes functionally important in many biological processes have been
discovered through the classical approach (Amsterdam and Hopkins,
2006; Brown and Hardisty, 2003; Cakar et al., 2012; Kurowska et al.,
2011; Mullins and Kang, 2001; Sauer, 2001). In most cases, moreover,
discoveries through the classical approach were comprehensively
unpredictable and even led to deeper understanding of the biological
complexity of various organisms. While the evolutionary strategy
was effective for improving the performance of the target strain, accumu-
lation of unwanted mutations may culminate into detrimental effects on
the strain. Perhaps most importantly, it is difficult to trace the genes
underlying the altered phenotype. Proteomic or transcriptomic com-
parisons have been implemented to infer gene targets responsible for
a phenotype (Blazeck and Alper, 2010; Kim et al., 2012a; Lee et al.,
2003). Quantitative trait loci (QTL) mapping, genetic linkage mapping,
or other high-throughput technologies have also been employed to
discover genes responsible for the phenotypes caused by spontaneous
or induced mutations (Deutschbauer and Davis, 2005; Madsen et al.,
2011; Perlstein et al., 2006, 2007; Yang et al., 2001). While evolving
rapidly, these analytical and genetic techniques are extensively time-
consuming and have shown limited success in metabolic engineering.
Next-generation sequencing (NGS) has recently gained wide attention
for its ability to conveniently trace the genetic basis of the mutant phe-
notype (Bro and Nielsen, 2004; Conrad et al., 2011; Hong et al., 2011; Le
Crom et al., 2009; Lee and Palsson, 2010). However, genome sequence
assembly via NGS requires a sequenced reference; genome sequence
analysis without a reference genome is still challenging and rarely
reported (You et al., 2011). Further validation is also necessary to dis-
cover the mutation responsible for phenotype improvement. Using
NGS, it is technically difficult to prove the linkage between mutation(s)
and phenotype if there are multiple mutations. In summary, an evolu-
tionary strategy is an efficient tool to improve a strain, but it is still
challenging to identify any gene(s) responsible for the phenotypes.
The evolutionary strategy can be included in an inverse metabolic
engineering (IME) approach, also known as a combinatorial approach,
according to the original definition by Bailey; “the elucidation of a met-
abolic engineering strategy by: first, identifying, constructing, or calcu-
lating a desired phenotype; second, determining the genetic or the
particular environmental factors conferring the phenotype; and third,
endowing the phenotype into another strain or organism by directed
genetic or environmental manipulation” (Bailey et al., 2002). However,
the evolutionary strategy has a limitation for identifying the target gene
to confer the phenotype as mentioned before. In contrast to the evolu-
tionary strategy, exploitation of genetic libraries capable of perturbing ge-
netic or metabolic circuits at various levels allows discovery of unexplored
genes responsible for a particular phenotype of interest. In addition,
the gene target identified through genetic libraries is conveniently
 H.J. Kim et al. / Biotechnology Advances 31 (2013) 976–985 979

transferable to another strain to confirm its function even under dif-
ferent strain backgrounds. There are three IME approaches using
combinatorial libraries (Fig. 2). The first approach is a genome scanning
method through knockout or overexpression libraries to identify a
novel target conferring a desired phenotype. The second approach is
to modulate expression levels of key genes in metabolic pathways by
synthetic promoters (Alper et al., 2005a; Blazeck et al., 2011; Hartner
et al., 2008; Nevoigt et al., 2006) or tunable intergenic region (TIGR)
libraries (Pfleger et al., 2006). The third approach utilizes global tran-
scription machinery engineering (gTME) or artificial transcription
factor (ATF) libraries to engineer global transcriptome. Selection of an
adequate library containing enough coverage and development of an
efficient screening tool to isolate desired strains are key steps for the
successful combinatorial library approach (Gill et al., 2002; Lynch
et al., 2007; Michener and Smolke, 2012; Tyo et al., 2006). Library
construction and mutant screening steps are typically followed by
identification and characterization of the obtained mutants. Finally,
transfer of the identified genetic perturbations into another strain
is performed for confirmation.
3. Single gene overexpression and deletion libraries: mines of
new discovery
Despite strong understanding of biochemical processes, there may
be many unexplored genetic targets leading current research into a
new direction. The rational metabolic engineering approach focuses
on altering metabolic fluxes as intended using analytical tools such
as stoichiometric models and algorithms. Due to this focus, the rational
approach tends to rely on metabolic fluxes determined by enzymes
rather than to consider regulatory factors influencing metabolic flux
indirectly (e.g. transcriptional and translational regulators and genes in-
volved in modification or proteolysis of key proteins of metabolic fluxes).
However, genome-wide scanning of target genes using overexpression
(Fig. 2A) or knockout libraries (Fig. 2B) can reveal novel and otherwise
undiscovered genes affecting the production of a target molecule indi-
rectly. Genome-wide scanning of target genes can be more efficient for
improving a particular phenotype when unoptimized heterologous path-
ways are introduced or cells are cultured under unusual environmental
conditions. These cases are frequently observed when we attempt to

Transposon vector Introduction of

Genomic DNA mutation Key enzyme

X
Promoter
Generation of Establishment of Transposition
genomic DNA transposon
fragments mutant library

~ ~
~
X X ~ ~
Transformation ~
~ ~
X X ~
~
Transformation
Promoter library of key Different levels of
enzyme(s)
Genomic DNA library B transcripts
construction A C
F D Various expression levels of TIGR structure in a
E different enzymes synthetic operon
Rapid and Phenotypical
continuous ~ ~
~
genome
perturbations ~
~ ~
~
engineering ~
ssDNA oligos ~
~ ~
~
~ ~
~

Transformation
Transformation

Random Error-prone
shuffling PCR
TF RP TF

Zinc finger TIGR library

Combinatorially
motifs
optimized genome ATF or gTME libraries

Fig. 2. Simplistic diagrams representing the six major methods of inverse metabolic engineering presented in this paper. A) Genome-wide perturbation library. DNA fragments from
the whole genome are introduced into plasmids to generate a genomic DNA library. Each transformant contains a DNA fragment that may improve the production of a target mol-
ecule. B) Knockout library. Random insertion of a transposon into the genome leads to generation of a knockout library (transposon mutant library). C) Promoter library. Different
strength promoter(s) of key enzyme(s) result in transcript level alterations of strains. D) Tunable intergenic region (TIGR) library. Different RNA structures in a synthetic operon
lead to various expression levels of key enzymes. E) Global transcription machinery engineering (gTME) library and artificial transcription factor (ATF) library. A gTME library is
constructed by mutagenesis of a transcription factor. Transformants containing mutant transcription factors show varying phenotypes. An ATF library is generated by random shuffling
of zinc finger motifs. Introduction of both libraries leads to phenotype change in transformants. TF, transcription factor; RP, RNA polymerase. F) Multiplexed automated genome engineer-
ing (MAGE). A synthetic oligonucleotide library is introduced into cells. An automated device for a MAGE method produces beneficial phenotypes of the cells by recombination. Rapid and
continuous genome engineering forces combinatorial pathway optimization in the strain of interest.
980 H.J. Kim et al. / Biotechnology Advances 31 (2013) 976–985
produce biofuels and biochemicals (for example, ethanol production
from cellulosic biomass). Because biological pathways are often opti-
mized for a favored environment, overexpression or deletion of genes
may not alter metabolic fluxes. However, the introduction of heterolo-
gous genes or cultivation of the engineered organisms under unusual
environments for industrial applications may imbalance metabolic fluxes,
which negatively affects product formation (Lee et al., 2012). Gene tar-
gets reducing the imbalances of the metabolic fluxes can be identified
using overexpression or knockout libraries.
3.1. Examples of combinatorial genetic perturbations for producing fuels
and chemicals
Overexpression or knockout libraries covering the whole genome
have been proven to efficiently identify uncharacterized target genes
to improve the production of biofuels and biochemicals (Table 1). For
example, lycopene production by the engineered E. coli was signifi-
cantly improved by manipulating gene targets identified through
knockout and overexpression library screening (Alper et al., 2005a,
2005b, 2005c; Jin and Stephanopoulos, 2007). Specifically, novel
gene targets (yjiD, rssB and yjfP) improving lycopene production in
E. coli were identified through transposon-based insertional mutagenesis
(Alper et al., 2005c). Insertions of transposon were observed inside open
reading frames of rssB and yjfP, but transposon was inserted between the
open reading frame and a promoter region in the case of yjiD, suggesting
transcriptional perturbation rather than knockout of yjiD is a reason for
improved lycopene production. Moreover, these combinatorially iden-
tified targets were combined with the knockout targets (gdhA, ace,
gpmB and fdhF) identified systematically using stoichiometric modeling
(Alper et al., 2005b). Exploration of a metabolic landscape determined
by the identified targets through both systematic and combinatorial
searches led to the construction of an engineered strain accumulating
impressive amounts (18,000–23,000 ppm, 18–23 mg lycopene/g cell)
of lycopene (Alper et al., 2005c). Overexpression targets improving
lycopene production in E. coli were also identified using E. coli genomic
libraries. Novel gene targets (yjiD, ycgW, yhbL, purDH and yggT) as well
as the previously known targets (dxs, idi, rpoS and appY) for improving
lycopene production were identified. Moreover, exhaustive combina-
tions of the knockout and overexpression targets also yielded an
engineered E. coli accumulating 16,000 ppm (16 mg lycopene/g cell)
(Jin and Stephanopoulos, 2007). Importantly, the identified gene targets
capable of improving lycopene production had unknown functions,
making these genes difficult to be discovered through the rational
approach. Lee et al. (2011) also identified genes that improved galac-
tose utilization in S. cerevisiae through genome-wide perturbation li-
braries. Among these discovered genes, SNR84 and a truncated TUP1
had not yet been reported. Overexpression of SNR84 and a truncated
TUP1 showed improvements similar to the strains overexpressing
PGM2 coding for phosphoglucomutase, which is known to be a limiting
enzyme for galactose fermentation (Lee et al., 2011).
Screening of transformants with genomic libraries containing ran-
domly fragmented DNAs has led to unexpected findings in addition to
finding overexpression targets. First, introduction of extra copies of
endogenous genes or heterologous genes from other organisms is
feasible using a genomic library (Jin et al., 2005; Shahsavarani et al.,
2012). For example, Jin et al. (2005) introduced genomic libraries of
Scheffersomyces stipitis (formerly Pichia stipitis) into S. cerevisiae for
the improvement of xylose assimilation and ethanol production.
Because S. cerevisiae is unable to natively ferment xylose, key genes
XYL1, XYL2 and XYL3 encoding xylose reductase, xylitol dehydroge-
nase and xylulokinase, respectively, for the xylose metabolic pathway
from S. stipitis were introduced into S. cerevisiae. Sequential introduc-
tion of genomic libraries of S. stipitis into S. cerevisiae resulted in a
100% increase in the growth rate and a 70% increase in ethanol produc-
tion in a S. cerevisiae strain overexpressing the S. stipitis TAL1 gene
(Table 1). Additional introduction of a heterologous genomic library
into an engineered strain with an imported pathway can be attempted
to identify unexpected synergistic interactions between the newly in-
troduced gene(s) and the imported pathway. However, introduction
of a heterologous genomic library could be applicable only when the
heterologous genes from the library are compatible with the host strain.
If RNA polymerase, spliceosome or other genetic components are un-
able to operate properly on the heterologous genes, the introduction
of a heterologous genomic library may not be operational. In this case,
an alternative method, such as overexpression of heterologous cDNA,
can be considered. A genome-wide perturbation library is also advanta-
geous due to its ability to inactivate a gene by random DNA fragmenta-
tion, which often truncates certain genes. As mentioned previously, Lee
et al. (2011) discovered that galactose fermentation was significantly
improved through overexpression of truncated TUP1 proteins, a tran-
scriptional co-repressor of GAL genes (GAL1 and GAL4). A yeast strain
overexpressing truncated TUP1 gene led to a 250% increase in both
galactose consumption rate and ethanol productivity compared to the
control strain (Table 1). Interestingly, the deletion of TUP1 increased
biomass, but decreased ethanol production. This result indicated that
deletion of TUP1 was not beneficial for ethanol production using galac-
tose fermentation (Lee et al., 2011). The truncated form of TUP1 appeared
to compete with the wild type TUP1 protein during galactose utilization.
This competition between the truncated and the wild type TUP1 may
have prevented the assembly of the functional Tup1-Ssn6-Mig1 complex,
leading to derepression of the GAL gene that is beneficial to galactose fer-
mentation. However, the beneficial effect by genomic DNA fragmentation
is rare since the main purpose of utilization of a genomic library is to alter
the phenotype by overexpressing a full-length gene. Moreover, it is
challenging to construct an inactive truncated form for an entire genome
using a genomic library. It may be interesting to establish a DNA fragmen-
tation library of a certain important regulator(s) serving as a critical
component of the process for producing biofuels and biochemicals.
Another important advantage of the genome-wide perturbation library
is the ability to unearth key components in metabolic circuits. Kim et al.
(2012b) introduced a S. cerevisiae genomic library into a yeast strain
containing S. stipitis XYL1, XYL2 and XYL3 and observed that efficient
xylose-fermenting transformants contained an additional copy of the
XYL2 gene. It appeared that homologous recombination occurred be-
tween the library plasmid and the XYL2 gene. The transformant harboring
additional XYL2 among three enzymes involved in the xylose assimilation
pathway became more dominant through serial cultures due to more
efficient xylose consumption. Overexpression of XYL2 led to an increase
of ethanol yield (0.1 to 0.3 g ethanol/g xylose) and a decrease of xylitol
accumulation (0.4 to 0.1 g xylitol/g xylose). Likewise, it may be possible
to discover additional key genes necessary to overexpress through the
genome-wide perturbation library.
3.2. Considerations for constructing genetic perturbation libraries
The coverage of the genomic library can be validated using minimal
media screening, PCR amplification or other methods (Hong et al.,
2010; Jin et al., 2005). It is necessary to screen a large enough number
of transformants to cover the entire genome (Jones et al., 2008). How-
ever, screening and evaluation processes with sufficient numbers of
transformants to completely cover the genome is labor-intensive and
challenging when eukaryotic organisms that have a large genome size
are used for constructing libraries. To facilitate rapid screening and
evaluation processes, a systematic yeast overexpression library (also
known as a tiling library consisting of 1588 plasmids) was constructed,
encompassing the whole genome of S. cerevisiae (97.2% of the 12
megabase yeast genome). As the average insert size is ~8.7 kilobases,
the inserts have a larger number of open reading frames (ORFs) than
general genomic library inserts (Jones et al., 2008). While the tiling
library allows rapid and convenient screening with higher genome
coverage, it might be laborious to determine causal gene(s) responsible
to the phenotype by interrogating several combinations of genes within
 H.J. Kim et al. / Biotechnology Advances 31 (2013) 976–985
the insert. Similarly, construction of a tiling library covering the whole
genome of other eukaryotic microorganisms could be valuable to dis-
cover genes for producing biofuels and biochemicals.
As a complementary approach to overexpression libraries, random
knockout libraries using an insertional mutagenesis such as a trans-
poson have also been employed to improve the production of various
chemicals (Alper and Stephanopoulos, 2008; Reeves and Weber, 2012;
Tang et al., 2009; Tannler et al., 2008; Tyo et al., 2009). Elimination of
competing pathways or byproduct formation pathways via knockout
mutations could maximize yield of target biofuels and biochemicals.
The knockout library method, however, limits examination of the effect
of essential genes on the process since deletion of the essential genes
leads to inviability of the strain. Still, a gene knockdown library using
RNA interference (RNAi) or antisense can be used as an alternative
method to circumvent the inviability problem of the essential genes.
Because these methods are able to significantly reduce the expression
level of the transcript, an effect of knocking-out the gene without killing
the strain is apparent (De Backer et al., 2001). To use the RNAi or antisense
libraries, endogenous RNAi machinery such as Dicer, RNA-induced silenc-
ing complex and Argonaute proteins is necessary. Bacteria and most
Saccharomyces spp. completely lack or have incomplete RNAi machinery;
therefore RNAi or antisense libraries are not applicable (Drinnenberg
et al., 2009; Harrison et al., 2009). Although there are few reports
concerning RNAi or antisense libraries for the production of biofuels
and biochemicals thus far, the gene knockdown library can be exploited
when using an applicable host microbe.
4. Coordination of single or multi gene(s) expression
Based on prior knowledge on genetic circuits controlling meta-
bolic pathways, it can be determined which transporter or enzyme
is rate-controlling. While overexpression of genes coding for the
controlling transporter or enzyme might lead to enhanced produc-
tion of a target molecule, excessive and monotonous overexpression
of the genes could be detrimental to the microbes. Therefore, opti-
mized expression levels of the target genes are necessary to achieve
maximum production levels of a target product. There are two methods
for optimizing expression levels of target genes through combinatorial
libraries: (i) modulation of transcription via modification of promoter
and/or terminator and (ii) perturbations of mRNA stability or translation
efficiency via engineering RNA-based control modules such as mRNA
secondary structures, RNase cleavage sites and ribosome binding site
sequestering sequences.
4.1. Modulation of transcription via promoter modification
Promoter libraries (Fig. 2C) have been employed to optimize tran-
scription levels of target genes in several prokaryotes such as E. coli,
Lactococcus lactis and Lactobacillus plantarum (Alper et al., 2005a;
Jensen and Hammer, 1998a; b; Rud et al., 2006; Solem and Jensen,
2002). A mutation library of the promoter region between the −35
and −10 consensus sequences in L. lactis was screened and a mutant
promoter exhibiting a 400-fold increase of β-galactosidase as compared
to a parental promoter was isolated (Jensen and Hammer, 1998b). Alper
et al. (2005a) developed a method to quantify the strength of promoter
libraries by monitoring mRNA and protein levels of a reporter gene
using real-time PCR and flow cytometry. After characterization of the
promoter library, strong promoters were selected and introduced in
front of the dxs gene in a recombinant E. coli strain overexpressing
ispFD and idi for determining optimal expression levels of dxs for
improved lycopene production (Alper et al., 2005a). Various promoter
libraries were also generated to modulate target gene expression in
eukaryotes such as S. cerevisiae, Pichia pastoris and Yarrowia lipolytica
(Blazeck et al., 2011; Hartner et al., 2008; Nevoigt et al., 2006; Qin
et al., 2011). The replacement of a native promoter with mutant TEF1
promoters was performed to enhance expression levels of a target
981
gene in S. cerevisiae (Nevoigt et al., 2006). The results of real-time PCR
analysis of the target mRNA indicated the mutant promoters activity
spanning 8% and 120% of the activity of the unmutated TEF1 promoter.
Hartner et al. (2008) generated a library of mutant promoters by mod-
ulating putative transcription factor binding sites within the P. pastoris
AOX1 promoter to improve the production of a target protein. When
GFP (green fluorescent protein) was used for measuring the strengths
of the mutant promoters, a wide range of promoter activity (6%–160%
of the wild type promoter activity) was observed. In the high cell densi-
ty fed-batch fermentation, target protein (HRP) production under the
control of the selected mutant AOX1 promoter increased 129% as com-
pared to when the wild type AOX1 promoter was used. In Y. lipolytica,
Blazeck et al. (2011) created a synthetic library consisting of both an
enhancer element and a core promoter element. The synthetic library
exhibited the strongest promoter activity ever reported for Y. lipolytica.
A fluorescence-based assay using GFP indicated that the strongest
mutant promoter had 8-fold-higher fluorescence levels than those by
typically used endogenous promoters. Du et al. (2012) attempted to
balance the flux of target metabolic pathways in S. cerevisiae by combi-
natorial assembly of genes coding for improved cellobiose or xylose fer-
mentation under the control of diversified promoters. To this end, the
ten mutant promoters showing various strengths generated through
error-prone PCR for the three constitutive yeast promoters (PDC1,
ENO2 and TEF1) were combined with genes coding for xylose or cellobi-
ose utilizing enzymes. Growth-based screening was performed to select
desired mutants containing high efficient xylose or cellobiose path-
ways. The optimized pathways by the combinatorial assembly offered
both a xylose utilizing pathway with near-highest efficiency and a cello-
biose utilizing pathway with highest efficiency that were ever reported
in literature for S. cerevisiae strains (Du et al., 2012).
4.2. Modulation of transcription via engineering RNA-based
control modules
In addition to the promoter libraries, synthetic libraries of RNA-based
control modules capable of changing mRNA stability and translational
efficiency have been employed to optimize expression levels. Tunable
intergenic region (TIGR, Fig. 2D) libraries were introduced into a
synthetic operon to optimize expression levels of key genes involved
in artemisinin production (Pfleger et al., 2006). Specifically, to
manufacture amorpha-4,11-diene, a precursor to the antimalarial
drug artemisinin, three genes in the mevalonate pathway, atoB
(acetoacetyl-CoA thiolase), HMGS (HMG-CoA synthase) and tHMGR
(truncated HMG-CoA reductase) were clustered into one synthetic
operon containing varied TIGRs among the three genes. Enrichment
by transforming the TIGR library into E. coli DP5, a strain engineered
to be auxotrophic for mevalonate, and a fluorescence-based screening
yielded an engineered E. coli accumulating 7-fold higher mevalonate
(Pfleger et al., 2006). A similar synthetic library of an RNA-based control
module was utilized in eukaryotic cells as well. Babiskin and Smolke
(2011) appended an RNase cleavage site into a target gene to change
the stability of the corresponding mRNA in S. cerevisiae. RNase cleavage
of 3′ UTR of mRNA decreases the stability of the RNA transcript, leading
to the reduced enzyme production. Therefore, a synthetic library of
RNase cleavage sites with different RNA structures might enable to
modulate transcript and protein levels through variations in an RNase
processing efficiency (Babiskin and Smolke, 2011). A synthetic library
of the ERG9 expression cassette with various RNase cleavage sites was
generated to tune the expression level of Erg9p for the enhanced pro-
duction of ergosterol, a starting material for the synthesis of various
tetracyclic triterpenoid derivatives, in S. cerevisiae. As the Erg9 is a key
controlling enzyme of the metabolic flux partition between ergosterol
and squalene production, mutations in an RNase cleavage site of the
ERG9 gene could increase ergosterol production. While there have
been few attempts using synthetic libraries based on RNA-based control
982 H.J. Kim et al. / Biotechnology Advances 31 (2013) 976–985
modules, numerous unexplored applications related to biofuel and bio-
chemical production exist.
5. Orchestration of global transcriptome through simultaneous
perturbation of multiple gene targets
5.1. Global transcription machinery engineering (gTME)
Although deletion or overexpression of a single gene might affect
the production of a target molecule significantly, in many cases, ma-
nipulation of multiple genes is necessary for improved production
of the target molecule. However, identification of optimal combinations
of the multiple genes is difficult and time-consuming. This difficulty was
cleverly overcome by gTME (global transcription machinery engineer-
ing, Fig. 2E) (Alper et al., 2006). Instead of investigating massive combi-
nations of multiple genes responsible for multigenic phenotypes, such
as tolerance to ethanol or high temperature, transcriptional machinery
was engineered to improve the multigenic phenotypes. It was anticipat-
ed that mutant transcriptional machinery might modulate transcription
levels of multiple genes simultaneously so that the multigenic phe-
notype can be maximized through mutations in transcriptional ma-
chinery even without identifying and optimizing expression levels
of multiple target genes (Alper et al., 2006). Specifically, as mutations
of a TATA-binding protein can affect RNA polymerase preference and
promoter specificity (Schultz et al., 1992), multiple mutations were
introduced into a TATA-binding protein (SPT15) coding for a RNA po-
lymerase II general transcription factor using error-prone PCR (Alper
et al., 2006). The mutated SPT15 library (the total library size was
about 10 5) was introduced into yeast in order to select the ethanol/
glucose tolerant phenotypes. Mutant yeast strains containing the mu-
tated SPT15 library were sub-cultured by increasing selection pressure.
After growth-based screening, mutant strains with improved glucose
and ethanol tolerance were isolated. The best strain exhibited a 69% in-
crease of volumetric ethanol productivity.
As compared to the complex transcriptional machinery of eukaryot-
ic cells, prokaryotic cells have rather simple transcriptional machinery
governed by several sigma factors. Among the sigma factors, a primary
sigma factor (rpoD) was selected as a target gene to elicit global pertur-
bation of gene expression (Alper and Stephanopoulos, 2007). A ran-
domized mutant library of rpoD generated by error-prone PCR was
introduced into E. coli for improved tolerance to ethanol and lycopene
production. After growth-based selection or visual screening of the
transformants with the mutant library, various rpoD mutants eliciting
simultaneous perturbation of multiple genes were isolated. These
results suggest that gTME can be applied to prokaryotic cells as
well. Zhuo et al. (2010) executed a similar study to improve the pro-
duction of avermectin in Streptomyces avermitilis. A gTME library of
mutagenized sigma factor hrdB was introduced in S. avermitilis.
High-throughput screening using microplates was employed to se-
lect desired strains. Production of avermectin from selected strains
was confirmed in flask fermentation. The best transformant among
the strains expressing evolved hrdB produce 6.38 g∕L of avermectin
(50% yield improvement).
Securing a transcription factor library capable of changing gene
expression levels of putative target genes simultaneously is critical
for successful gTME. However, it is laborious and difficult to create a
gTME library covering the exhaustive combination of mutations in a
targeted transcription factor. Theoretical and practical aspects to be
considered for generating an effective gTME library were reviewed
previously (Lam et al., 2010).
5.2. Artificial transcription factor (ATF) libraries
Similarly to a gTME library, artificial transcription factor (ATF, Fig. 2E)
libraries have been employed to alter phenotypes of cells under various
environmental conditions. Eukaryotic transcriptional factors consist of a
DNA binding domain and transcriptional activating/repressing domain
(Ansari and Mapp, 2002). This modular nature of the transcriptional fac-
tor can be exploited for generating ATFs that contain programmable DNA
binding domains. Park et al. (2003) constructed an ATF library consisting
of the zinc-finger (ZF) domains fused to either the Gal4 activation domain
or the Ume6 repression domain in yeast. Random shuffling of three or
four ZF domains capable of binding three unique nucleotides resulted in
diverse transcription factors possessing different affinities for binding
specific DNA sequences. As a result, various ATFs facilitating diverse tran-
scription levels of multiple genes can be constructed. Mutant yeast cells
possessing the ATF library improved resistant phenotypes against heat
shock (25-fold increase in the thermotolerance phenotype under heat
treatment at 52 °C for 2 h), osmotic pressure (100-fold increase in the
osmotolerance phenotype under 100 mM LiCl) and anti-fungal agents
(10-fold to 100-fold increase in ketoconazole resistant phenotype)
could be screened (Park et al., 2003). Similar ATF libraries were also
employed in animal cells to show its utility in mammalian cell culture
systems (Park et al., 2003). The mouse neuroblastoma cell line (Neuro2A)
expressing ATFs composed of ZFs and a transcription activation (p65) or
repression (KRAB) domain displayed diverse neuronal differentiation
capabilities. In a further study, an ATF library containing ZF domain and
a transcription activation (V16) or repression (KRAB) domain were intro-
duced into a Chinese hamster ovary cell to boost monoclonal antibody
(mAb-72) production (Kwon et al., 2006). Among 2000 animal cells,
the best strain exhibited a 10-fold increase of monoclonal antibody
production.
An ATF library was also employed to obtain desired phenotypes in
prokaryotic cells. For example, Park et al. (2005) utilized an ATF
library with only DNA binding domains to identify a gene involved
in the thermotolerance of E. coli. ubiX involved in ubiquinone biosyn-
thesis gene was identified as a knockout target through elucidating
the binding site of the identified AFT conferring thermotolerance in
E. coli (Park et al., 2005). Likewise, Lee et al. (2008) created an ATF
library that is composed of a ZF DNA-binding protein fused to an
E. coli CRP (cyclic AMP receptor protein) effector domain. As a CRP is a
global transcription factor capable of binding to more than 200
promoters in E. coli, utilization of a CRP effector domain may lead to
diverse transcription levels of multiple genes in the cells. Random-
shuffling of 40 different types of ZFs resulted in the generation of
more than 6.4 × 104 ATFs. Introduction of the ATF library into E. coli
allowed for obtaining the prokaryotic cells showing resistant pheno-
types against heat shock (55 °C for 2 h), osmotic pressure (0.6 M
NaCl) and cold shock (15 °C) treatments (Lee et al., 2008), the condi-
tions that inhibit the growth of wild type E. coli severely or completely.
Thermotolerant, osmotolerant and cold-tolerant ATF transformants
(50, 150 and 13 transformants respectively) were selected and
characterized. The best thermotolerant transformant selected from
flask fermentation experiments on 50 °C was used for further iden-
tification of genes involved in the thermotolerance phenotype.
Microarray results indicated that a total of 202 genes in the best
thermotolerant transformant were significantly altered (more than
4-fold change compared to the control) in the heat treatment.
Therefore, the identified genes can be used for future application
of thermotolerance.
Global regulation of transcriptome by a gTME or ATF library has
endowed phenotypical changes to host microbes resulting in resistance
against severe environments such as osmotic or temperature stresses
rather than elevating yield and productivity. Strategies using gTME and
ATF libraries may improve tolerance of strains against fermentation inhib-
itors commonly found in cellulosic hydrolysates, such as 2-furaldehyde
and 5-hydroxymethyl-2-furfural, in cellulosic hydrolysates (Brethauer
and Wyman, 2010; Park et al., 2011). Therefore, tolerance of desired
strains against fermentation inhibitors can be beneficial in terms of
price competitiveness of cellulosic biofuels as it may avoid complicated
separation procedures that would otherwise be required to remove
inhibitors.
 H.J. Kim et al. / Biotechnology Advances 31 (2013) 976–985
5.3. Multiplexed automated genome engineering (MAGE)
Recently, multiplexed automated genome engineering (MAGE,
Fig. 2F) was demonstrated for efficient and simultaneous engineering
of multiple genes involved in a target metabolic pathway (Isaacs et al.,
2011; Wang et al., 2009, 2012). The repeated introduction of a pool of
targeting oligonucleotides using automated devices can generate di-
verse desired mutations into the cell continuously and rapidly and can
enrich beneficial phenotypes. The λ-Red recombination system that
promotes single-strand annealing is exploited to replace desired gene
loci with synthetic oligonucleotides with high frequencies. To verify fea-
sibility of a MAGE method, 24 target genes, previously known as critical
genes for lycopene production, in the 1-deoxy-D-xylulose-5-phosphate
biosynthesis pathway in E. coli were engineered by a MAGE method
(Wang et al., 2009). Synthetic oligonucleotides containing degenerate
ribosome binding site sequences were used to replace the ribosome
binding sites of the target genes. The MAGE method could generate
more than 4.3 × 10 9 combinatorial genomic variants per day. After
5–35 cycles of MAGE, ~ 10 5 colonies were isolated based on colori-
metric screen. Among them, two mutant strains showed a 5-fold in-
crease in lycopene production (~ 9000 ppm, 9 mg lycopene/g cell)
compared to the parental strain (Wang et al., 2009). The MAGE method
was also utilized to improve indigo production in E. coli (Wang et al.,
2012). Multiple T7 promoters were simultaneously inserted into 12
genomic operons to optimize the indigo and indirubin biosynthesis.
The best strain among variants containing T7 promoter insert combina-
tions exhibited a 62% increase in indigo production (8.6 mg/g dry cell
weight of indigo) than the parental strain (Wang et al., 2012). As a com-
plementary method, trackable multiplex recombineering (TRMR) was
developed to engineer thousands of genes in a genome simultaneously
and to identify the mutations responsible for a phenotype of interest
rapidly (Warner et al., 2010). The TRMR strategy allowed for discover-
ing the genes responsible for the growth of E. coli in four different
conditions (salicin, D-fucose, methylglyoxal and valine) in which wild
type E. coli were typically unable to grow. Sequentially, individual
colonies (83 total) were selected and the genes responsible for the
growth on the inhibitors were identified using barcode sequences
and microarrays. The TRMR method identified the most critical
genes for growth of E. coli on each inhibitor as hns for salicin, xylA
for D-fucose, sodC for methylglyoxal and ilvN for valine respectively.
The TRMR strategy also allowed simultaneous mapping of thousands
of genes affecting the growth of E. coli in the minimal and rich medium
as well as lower (15%–17%) and higher (18%–20%) concentrations of
lignocellulosic hydrolysate within 1 week (Warner et al., 2010). Unfor-
tunately, the MAGE and TRMR methods are only applicable to the
organism whose genome sequence and single-strand recombination
mechanisms are known. Relative difficulty to transfer acquired mutation
into a new strain is another obstacle of the MAGE and TRMR methods, as
compared to other library methods.
While these combinatorial perturbations of genetic elements
using various methods would allow exploration of metabolic spaces
that cannot be realized via rational perturbation methods, these ap-
proaches are currently limited due to a lack of general screening
methods for selecting the intended phenotypes caused by combinatorial
libraries. Rapid advances in developing high-throughput screening will
complement the approach for combinatorial genetic perturbation for
producing biofuels and biochemicals (Macarrón and Hertzberg, 2011;
Michael et al., 2008).
6. Conclusions and future directions
Increasing demand for biofuels to replace fossil fuels has been a
strong driving force advancing methodologies of metabolic engineering.
The rational approach based on prior information is still a major meth-
odology of strain improvement. However, a combinatorial approach is
providing unprecedented critical information on how to improve target
983
strains toward the desired phenotypes. The importance of a combinato-
rial approach may increase as the amount of available information for
rational design will likely soon be drained. Therefore, a combinatorial
approach could serve as a great resource of information to generate
hypotheses for the rational approach.
A hybrid approach through fusing different kinds of combinatorial
libraries will confer versatility into microbial factories. As global gene
perturbation methods are amenable for engineering phenotypes deter-
mined by multiple genes, such as tolerance phenotypes, overexpression
or knockout libraries, which are effective for improving product yield
and productivity, can be combined with the global perturbation methods.
The hybrid approach will be ideal for implementation of microbial facto-
ries, allowing for significant breakthroughs in biofuel and biochemical
production.
Acknowledgment
This work was funded by the Energy Biosciences Institute.
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