Journal of Biotechnology 145 (2010) 341–349

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Review

Biotechnological applications of microbial proteomes

Mee-Jung Han a,∗ , Sang Yup Lee b , Seung-Tae Koh a , Sang-Gyun Noh a , Won Hee Han a
a
Department of Chemical and Biomolecular Engineering, Dongyang University, # 1 Gyochon-dong, Punggi-eup, Yeongju, Gyeongbuk 750-711, Republic of Korea
b
Departments of Chemical & Biomolecular Engineering (BK21 Program), Bio and Brain Engineering, and Biological Sciences, BioProcess Engineering Research Center, Bioinformatics
Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea

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

Article history: Advances in proteomic technologies have led to the creation of large-scale proteome databases that can
Received 18 August 2009 be used to elucidate invaluable information on the dynamics of the metabolic, signaling and regulatory
Received in revised form networks and to aid understanding of physiological changes. In particular, proteomics can have practical
21 November 2009
applications, for example, through the identification of proteins that may be potential targets for the
Accepted 23 December 2009
biotechnology industry, and through the extension of our understanding of the physiological action of
these proteins. In this review, we describe proteomic approaches for the discovery of targets that have
potential biotechnological applications. These targets include promoters, chaperones, soluble fusion part-
Keywords:
Biotechnology
ners, anchoring motifs, and excretion fusion partners. In addition, we discuss the potential applications
Microbial proteomics of proteomic techniques for the design of future bioprocesses and the optimization of existing ones. Suc-
Microorganisms cessful applications of proteomic information have proven to have enormous value for both scientific
Multi-omics and practical applications.
Two-dimensional gel electrophoresis © 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
2. Proteomic approaches for target discovery and new developments in biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
2.1. Identification of protein candidates employed as novel promoters from a whole cellular proteome under a given culture condition . . . 342
2.2. Identification of chaperones from a stress-responsive proteome to improve production performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
2.3. Identification of key enzymes in a regulatory or metabolic pathway from a whole cellular proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
2.4. Identification of fusion partners for enhanced solubility from highly soluble proteomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
2.5. Identification of anchoring motifs used for the cell surface display from membrane proteomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
2.6. Identification of fusion motifs from extracellular proteome for excretory protein production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
3. An integrated approach by proteomics combined with more than one of the multi-omic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

1. Introduction changes in protein synthesis, degradation rates, post-translational

modifications, and protein interactions, which enhances our
Proteomics has been widely used in a variety of basic and understanding and knowledge of physiological phenomena for a
applied microbiology research (Han and Lee, 2006; Josic and specific condition. In addition, proteomics can be used to dis-
Kovač, 2008). In contrast to conventional biochemical stud- cover novel targets (typically genes or proteins) to be engineered
ies that focus on a single protein or simple macromolecular for strain improvement and bioprocess development, which may
complexes, proteomics takes a broader, more comprehensive be even more important than the already known proteins and
and systematic approach to the investigation of biological sys- genes that are involved in a given condition. These potential
tems. Proteome analysis provides invaluable information about discoveries could lead to the construction of superior produc-
tion systems using rational modifications of biotransformation
and process development. Proteomics can be called, thus, a
∗ Corresponding author. Tel.: +82 54 630 1148; fax: +82 54 630 1275. discovery-based approach, since it is not constrained by prior
E-mail address: mjhan75@dyu.ac.kr (M.-J. Han). knowledge.

0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2009.12.018
342 M.-J. Han et al. / Journal of Biotechnology 145 (2010) 341–349
Recent advances in proteomic technologies have generated
large amounts of proteome data, which are used in fundamental
as well as applied research. For example, proteomic technologies,
such as mass spectrometry (MS) combined with stable isotopic tag-
ging methods (Zhou et al., 2002; Chakraborty and Regnier, 2002)
or an amine-reactive isobaric tagging method (iTRAQ; Ross et
al., 2004) method has increased the high-throughput analyses of
protein identification from biological samples and have allowed
for highly accurate protein quantification. These techniques are
applicable due to several advances, including the availability of
public genome and protein databases, the development of search
engines that are capable of exploiting these databases, and the
introduction of high-sensitivity, easy-to-use mass spectrometers.
Furthermore, in order to reduce complexity and increase sensi-
tivity for the detection of low-abundance proteins, proteomics
researchers have become increasingly interested in the combina-
tion of gel-independent technologies with subcellular fractionation
by n-dimensional chromatography (Huber et al., 2003; Taylor et
al., 2003). More detailed information on the technological and
methodological advances in gel-based and gel-independent pro-
teomic approaches and of predictive proteomics, which involve
two-dimensional gel electrophoresis (2-DE), MS, and computa-
tional tools can be found in a review article by Han and Lee (2006).
However, the method of 2-DE coupled with MS is still being widely
used for the proteomic analysis of microorganisms. In particular,
subcellular proteomics of microbial cells (Lopez-Campistrous et al.,
2005), which involves the analysis of subcellular compartments by
biochemical and physical fractionation, has been utilized due to
the additional benefits of reducing sample complexity, identifying
as many unique proteins as possible, particularly low-abundance
proteins specific to a particular compartment, localizing newly
discovered proteins to specific subcellular compartments, and pro-
viding a functional context for some proteins by associating them
with a distinct group of proteins in defined cellular compartments.
A more important benefit of microbial proteomic studies is
that it can be applied to develop and improve the various steps
that are part of industrial processes (Josic and Kovač, 2008). How-
ever, there is no a detailed review that how we can practically
use proteomic information that is contained in microorganisms to
biotechnological applications. In this review, we describe several
approaches to identify novel bacterial components, which are of
interest for their biotechnological applications, by proteomic anal-
yses even though there are a limited number of examples in the
literature to date. These approaches include the identification of
new and potentially useful promoters from whole cellular pro-
teomes, the characterization of fusion partners and motifs from
membrane or extracellular proteomes, and the enhancement of the
quality and quantity of recombinant proteins from highly soluble or
stress-responsive proteomes. In addition, we discuss the potential
applications of biotechnology for the design of future bioprocesses
and the optimization of existing ones. The successful application
of proteomic information is generating a revolution in the biotech-
nology industry.
2. Proteomic approaches for target discovery and new
developments in biotechnology
The rapid growth of proteomics, which was built upon the avail-
ability of sequenced bacterial genomes, has led to new approaches
in bacterial functional genomics. In studies on a variety of bacteria,
the combined technologies of genomics, proteomics, and bioin-
formatics have provided valuable tools for the study of complex
phenomena that are determined by the action of multiple genes and
pathways (Ideker et al., 2001; Joyce and Palsson, 2006). Recently,
combined transcriptome and proteome approaches have allowed
large-scale analysis of biological systems at the mRNA and protein
levels, providing us with unprecedentedly large amounts of infor-
mation that is useful in data-driven discovery (Baek et al., 2009;
Duy et al., 2007; Nguyen et al., 2007; Yoon et al., 2003). In par-
ticular, proteomic techniques may identify proteins that may be
potential targets for biotechnology and may help to extend our
understanding of the physiological actions of these proteins. Based
upon the needs of the researcher, the proteome or subproteome
profiling of an organism under a given condition can be utilized
to greatly enhance our understanding of complex biological pro-
cesses and further improve or newly develop production systems
in a variety of organisms. The workflow of this approach for exploit-
ing proteome data in biotechnological applications is illustrated
in Fig. 1. Prior to proteome analyses, the proteins of an organism
can be classified and divided into four fractions: (i) total proteins,
(ii) soluble proteins, (iii) membrane proteins from the inner and/or
outer membranes, and (iv) extracellular proteins. This approach
can facilitate the discovery of targets, such as promoters, chap-
erones, soluble fusion partners, anchoring motifs, and excretion
fusion partners, which can potentially be utilized for biotechno-
logical applications. These targets can be used to further improve
producing strains by recombinant DNA technology and this cycle
can be repeated until an optimal, custom-made strain is developed.
Proteomics has unquestionably led to new and valuable informa-
tion on microorganisms, and it will continue to be an important
source of information in the coming years.
2.1. Identification of protein candidates employed as novel
promoters from a whole cellular proteome under a given culture
condition
The development of recombinant DNA technology allowed the
large-scale production of medically and industrially useful pro-
teins, which were previously obtained only in small amounts
under natural conditions, by various genetically modified organ-
isms. Escherichia coli is the most widely used host for the production
of recombinant proteins, because it is such a well-characterized
system. There are three main factors that determine the produc-
tivity of a high-level protein expression system in host cells: (i)
promoter sequences that regulate the expression of target proteins,
(ii) signal sequences or fusion motifs that facilitate the localiza-
tion of target proteins within the cell, and (iii) plasmid origin.
Much research has focused on obtaining high-expression promot-
ers, motifs, and plasmids, and their identification and optimization
have been achieved either by ‘trial-and-error’ type approaches,
in which various genetic modifications were performed until a
desired objective was achieved, or by transposon mutagenesis.
Recently, this research area has been streamlined through the
use of genomic, transcriptomic, proteomic, and computational
tools, all of which have been used individually or in combination to
facilitate the development of ‘custom-made’ production systems
in microorganisms. Among these ‘-omic’ tools, proteomic studies
can be easily performed and have provided invaluable information
to rationally discover novel suitable promoters that are required
for specific expression systems, according to the needs of the
researcher. Differences in the upstream sequences of a fusion gene
can influence the efficiencies of its transcription and translation,
which results in variations in the amount of fusion protein that will
be expressed. In general, DNA sequences that are upstream of genes
that code for highly expressed proteins often contain strong pro-
moters. Therefore, proteins that are highly expressed under a given
condition can easily be isolated from high-abundance proteins on
2-D gels of the whole cellular proteome of an organism, followed
by the identification of their promoter regions based upon genomic
databases. In the magnetic bacterium Magnetospirillum magneticum
AMB-1, for example, proteome analysis was used to identify a
 M.-J. Han et al. / Journal of Biotechnology 145 (2010) 341–349 343

Fig. 1. Schematic illustration of various biotechnological applications within the subproteome maps of microorganisms. Once the research objective is set, microbial cells
can be cultured and sampled for proteomic profiling. During this process, protein samples can be prefractionated or differentially labeled for a better comparison of the
results. Proteome profiles can be obtained by gel-based and/or non-gel-based approaches. Predictive proteomic studies can also be performed to analyze the characteristics
of specific proteins. If possible, both gel- and non-gel-based approaches should be combined, since the two methods are complimentary, in order to maximize the total
number of proteins that are detected and identified. For biotechnological applications, the tremendous amount of proteome data can be utilized to select potential targets of
interest, including promoters, chaperones, solubility fusion partners, anchoring motifs, and excretion fusion partners, in order to create a custom-made strain. The various
genetic modifications of the parent strain, which are made using recombinant DNA technology, can be repeatedly performed until the desired objective is achieved.

few strong promoters, which were successfully used to develop
a novel, efficient expression system for the cell surface display
of a protein (Yoshino and Matsunaga, 2005). These new promot-
ers (Pmms16, Pmms24, Pmsp1, Pmsp2, and Pmsp3) showed higher
activity and efficiency to drive the expression of the luciferase gene
when compared to the magA promoter, which had previously been
used for the display of heterologous proteins on bacterial magnetic
particles. Specifically, the intensity of the luminescence that was
obtained using the msp3 promoter was 400-fold higher than that
obtained using the conventional magA promoter.
Another proteomic study involved the comparative analysis of
E. coli in the presence of glucose and oleic acid (Han et al., 2008b),
which isolated the promoter of the aldA gene as being specifi-
cally inducible by oleic acid but not glucose. The expression of the
reporter, green fluorescent protein (GFP), was much higher under
the control of the aldA promoter than under the control of the
widely used IPTG-inducible tac promoter. These studies demon-
strated that the choice of promoter would be a critical component
of a high-level expression system. In addition, these data indi-
cate that highly active promoters will be easy to obtain using DNA
sequences that are upstream of protein that were selected based
upon their high-expression levels and that, consequently, an effi-
cient protein expression system can be developed using the highly
active promoters. Therefore, proteomic profiles not only provide
invaluable information on the physiological status of the organism
under specific conditions, but also contribute to the improvement
of biotechnological applications. A novel promoter that is discov-
ered using proteomic data should display certain characteristics
with respect to the protein under its control in order to demonstrate
its suitability for use in biotechnological applications: (i) strong
and long-lasting expression that results in the accumulation of the
target protein such that it generally comprises at least 10–30% of
the total cellular proteins (Makrides, 1996); (ii) tightly controlled
expression under an appropriate inducer or a certain condition,
such as the phase of the cell cycle, the presence or absence of nutri-
ents, or particular stressors; and (iii) inducibility in a cost-effective
manner. In particular, the tight regulation of the promoter is essen-
tial for the synthesis of proteins that may be detrimental to the host
cell (Brown and Campbell, 1993; Doherty et al., 1993). For exam-
ple, the toxic rotavirus VP7 protein kills host cells, and therefore,
should be produced under tightly controlled environments (Emslie
et al., 1995).
2.2. Identification of chaperones from a stress-responsive
proteome to improve production performance
The most common type of proteomic study is a quantitative pro-
tein profile comparison of samples that were obtained under two or
more experimental conditions in order to examine the responses to
environmental stimuli or stresses. These studies can determine the
specific proteins that are up- or down-regulated in response to var-
ious chemical and physical stresses (Han and Lee, 2006; Neidhardt
and VanBogelen, 2000), such as heat (Han et al., 2008a; Rosen and
Ron, 2002), oxidative agents (Han et al., 2008a; Wolf et al., 2008;
Zeng et al., 2008), and hyperosmotic shock (Höper et al., 2006;
Soufi et al., 2009; Weber et al., 2006). These proteomic changes
are thought to act as protective mechanisms that lead to the elim-
ination of a stress agent and/or the repair of cellular damage, and
these cellular responses can differ widely according to the stress
imposed. Chaperones, which are often dramatically induced under
various genotypic and environmental conditions, play a central role
in the quality control of the proteome by interacting with, stabi-
lizing, and remodeling a wide range of non-native polypeptides
(Bukau et al., 2006). Several chaperones implicated in protein fold-
ing, such as DnaK, GroEL, and GroES (Goloubinoff et al., 1989; Murby
et al., 1991; Baneyx and Palumbo, 2003), have been used to improve
the yields of soluble recombinant proteins.
In a similar manner, small heat shock proteins (sHsps) could be
used to control the quality and quantity of recombinant proteins
(Kolaj et al., 2009). IbpA and IbpB, which are two representative
sHsps, were first identified by conventional biochemical techniques
as the major proteins that associate with the inclusion bodies (IBs)
of recombinant proteins produced in E. coli (Allen et al., 1992).
IbpA and IbpB facilitate the production of recombinant proteins
in E. coli and play important roles in protecting recombinant pro-
teins from degradation by cytoplasmic proteases (Han et al., 2004).
Amplification of the ibpA and/or ibpB genes enhanced the produc-
tion of recombinant proteins as IBs, whereas ibpAB gene knock-out
enhanced the secretory production of recombinant proteins in their
soluble forms (Han et al., 2004). In another study, ␣-glucosidase
344 M.-J. Han et al. / Journal of Biotechnology 145 (2010) 341–349
production was shown to be enhanced at elevated levels of IbpA
and IbpB and reduced in an ibpAB-negative mutant strain in a
temperature-dependent manner (LeThanh et al., 2005). In addi-
tion, IbpA and IbpB prevent ␣-glucosidase IBs from degradation in
a temperature-dependent manner. Taken together, these findings
suggest that manipulation of ibpAB gene expression might be an
efficient way to fine-tune the production of recombinant proteins
in E. coli. The beneficial effect of IbpAB on the production of recom-
binant proteins is, however, high when protease activity is high,
such that the benefits become diminished at moderate tempera-
tures (≤30 ◦ C) due to the decreased activities of cellular proteases
(Kuczyńska-Wiśnik et al., 2004). In order to achieve the maximum
desired effects, some experimental optimization studies need to be
performed, in which temperature, strain species, and/or environ-
mental conditions are examined, since these parameters affect the
expression of ibpAB and their subsequent effects.
The usefulness of sHsps has recently been extended, as they
have been shown to significantly enhance the performance of 2-
DE (Han et al., 2005). Protein spots in 2-D gels can often be lost
due to residual protease activity when using immobilized pH gra-
dient gels for isoelectric focusing. Three sHsps, including IbpA and
IbpB from E. coli and Hsp26 from Saccharomyces cerevisiae, were
shown to have the capability to protect proteins in vitro from pro-
teolytic degradation. The addition of sHsps during 2-DE of human
serum or whole cell extracts of bacteria (E. coli, Mannheimia suc-
cinciproducens), plant Arabidopsis thaliana, and human kidney cells
resulted in the detection of up to 50% more protein spots than those
obtainable with currently available protease inhibitors. As a result,
protein detection is enhanced, and many more protein spots are
visible than had been under previous conditions.
Another study monitored the physiological changes of recom-
binant E. coli during the secretory production of a recombinant
humanized antibody fragment by 2-DE (Aldor et al., 2005). Twenty-
five protein spots were altered in the control and production of
fermentation over a 72-h period, and, in particular, the synthesis
of the phage shock protein A (PspA) strongly correlated with the
synthesis of the recombinant product. Coexpression of the pspA
gene with a recombinant antibody fragment in E. coli significantly
improved the yield of this secreted biopharmaceutical. Therefore,
proteins with chaperone activity that are identified from proteome
profiling data can be used for the control of quality and quantity of
recombinant proteins.
2.3. Identification of key enzymes in a regulatory or metabolic
pathway from a whole cellular proteome
Microbial producing strains are often engineered to increase the
yield and productivity of native products synthesized, and to pro-
duce entirely novel bioproducts. At this point, an important thing to
be considered is the direct and indirect influences of the metabolic
pathways that supply the energy and precursors required for the
synthesis of bioproducts (Hoffmann and Rinas, 2001; Hoffmann et
al., 2002). Since bioproducts are formed by coordinated enzyme
reactions acting through metabolic pathways, understanding the
metabolism and regulation that occur during cell growth and prod-
uct formation is essential.
In industrial biotechnology, proteomic analysis has been applied
to directly monitor cellular changes that occur during the produc-
tion of heterologous proteins in microorganisms and to develop
efficient strains for the enhanced production of bioproducts (Aldor
et al., 2005; Han and Lee, 2006; Han et al., 2003; Hoffmann and
Rinas, 2001; Hoffmann et al., 2002; Jürgen et al., 2000; Raman et
al., 2005). Many of these proteomic studies have been carried out in
large-scale processes for industrial applications. For example, the
proteome profiling of E. coli during the high cell density cultivation
showed that the levels of many amino acid biosynthetic enzymes
decreased during the production of recombinant proteins (Han et
al., 2003; Jürgen et al., 2000; Raman et al., 2005). This finding gives
an explanation why the addition of amino acids or complex nitro-
gen sources enhanced production of recombinant proteins (Lee,
1996; Ramérez and Bentley, 1993). The results found by proteome
analysis provide that researchers can elucidate which molecules
supply the energy and building blocks or precursors, such as amino
acids and other metabolic intermediates, that are necessary for cell
function and product formation.
In addition, proteomic analysis can be used to identify rate-
controlling steps in metabolic pathways and develop a systematic
strategy for optimizing the relevant metabolic pathways. An pos-
sible approach for exploiting the proteome results to enhance
production of recombinant proteins (Han and Lee, 2003) is: (i)
identify potentially limiting enzymes in the metabolic and regu-
latory pathways, (ii) examine theoretically and/or experimentally
the possible pathway changes that can be achieved by manipulat-
ing the enzymes identified, (iii) select the targets to be manipulated,
(iv) examine the effect of this metabolic and cellular engineering,
and (v) repeat (ii) to (iv) until the researcher’ goal is achieved. Using
this approach, Han et al. (2003) reported protein profiling of recom-
binant E. coli during the overproduction of human leptin identified
cysK as a target gene, which led to the development of a success-
ful metabolic engineering strategy to increase the productivity of
leptin and other serine-rich proteins by co-expression of the cysK
gene. Thus, another effective use for proteomics is the identification
of candidates for the successful metabolic engineering to enhance
the formation of bioproducts. However, recent systemic investi-
gations by transcriptome, molecular biological, and mathematical
tools have become popular for the development of superior pro-
duction systems in microorganisms (Lee et al., 2007; Park et al.,
2007a).
2.4. Identification of fusion partners for enhanced solubility from
highly soluble proteomes
Although a favorable yield of a recombinant protein in bac-
teria can usually be accomplished using the recombinant DNA
technology illustrated above, obtaining the protein in a soluble,
biologically active form continues to be a major challenge. A
strategy for enhancing the solubility of aggregation-prone recom-
binant proteins is the use of a highly soluble fusion partner. Three
general soluble fusion partners, maltose-binding protein (MBP),
glutathione S-transferase (GST), and thioredoxin (TRX), have shown
good performance in enhancing the solubility of recombinant pro-
teins, but they are not universally effective at promoting the
solubility and folding of all proteins. Many novel, ‘custom-made’
fusion partners have been recently discovered by systematic inves-
tigations using proteomic studies. These partners were identified
as proteins that were significantly increased on 2-D gels from
bacterial cells under a few exogenous stresses, such as guanidine
hydrochloride (GdnHCl) or heat shock. Most of these proteomic
studies were performed by the Lee laboratory in Korea. These
fusion partners, including RpoS (RNA polymerase sigma factor)
(Park et al., 2008), Tsf (elongation factor Ts) (Han et al., 2007b),
Mdh (malate dehydrogenase) (Park et al., 2007b), SlyD (FKBP-type
peptidyl-prolyl cis-trans isomerase; PPIases) (Han et al., 2007c),
PotD (spermidine/putrescine-binding periplasmic protein), and Crr
(glucose-specific phosphotransferase (PTS) enzyme IIA compo-
nent) (Han et al., 2007a) were shown to be highly effective as
strong solubility enhancers for aggregation-prone heterologous
proteins in an E. coli expression system. Using these fusion part-
ners, many aggregation-prone heterologous proteins, including
human minipro-insulin (mp-INS), human epidermal growth factor
(EGF), human prepro-ghrelin (ppGRN), human interleukin-2 (hIL-
2), human activation induced cytidine deaminase (AID), human
 M.-J. Han et al. / Journal of Biotechnology 145 (2010) 341–349
glutamate decarboxylase (GAD448–585 ), Pseudomonas putida cuti-
nase (CUT), human ferritin light chain (hFTN-L), human granulocyte
colony-stimulating factor (G-CSF), and human cold autoinflamma-
tory syndrome 1 protein (NALP3) Nacht domain (NACHT), were
produced as biologically active fusion forms in E. coli cytoplasm.
These findings could prove to be useful for the production of other
biologically active and industrially valuable proteins or enzymes.
2.5. Identification of anchoring motifs used for the cell surface
display from membrane proteomes
Outer membrane proteins play an important role in the adap-
tation to changes in external environments due to their location
at the outermost edges of a cell. Many studies have shown that
the synthesis of outer membrane proteins would change when
bacteria were exposed to environmental changes involving vari-
ous stressors, such as iron limitation (Faraldo-Gómez and Sansom,
2003), drug resistance (Trias and Nikaido, 1990), osmotic stress
(Xu et al., 2005), antibiotics (Zhang et al., 2008; Lin et al., 2008),
or acid stress (Sato et al., 2000). Despite of the importance of
these membrane proteins, they are difficult to visualize on 2-
D gels of whole cell lysates due to their hydrophobic nature,
resulting that these proteins are underdetermined or remained
to be studied separately (Wu and Yates, 2003). For example,
the hydrophobic membrane proteins have known difficult to
solubilize with common solubilization agents such as urea, 3-
[(3-cholamidopropryl)dimethylammonio]-1-propanesulfonic acid
(CHAPS) and dithiothreitol (DTT). To overcome the difficulties
in membrane proteome analysis, a variety of analytical methods
including gel and non-gel-based approaches have been developed
in the past several years (Weiner and Li, 2008; Wu and Yates, 2003).
A detailed proteomic technologies and challenges in E. coli mem-
brane proteome studies can be found in a recent review article
(Weiner and Li, 2008). For example, a first isolation protocol, in
which increasing concentrations of sodium carbonate were used
to extract outer membrane proteins from E. coli, was originally
introduced by Molloy et al. (2000). This method led to the suc-
cessful identification of 21 out of 26 of the predicted integral
outer membrane proteins using 2-DE and matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) MS. Similarly,
Fountoulakis and Gasser (2003) identified 394 different gene prod-
ucts using the sodium carbonate method (Molloy et al., 2000) to
create the largest database of E. coli membrane proteins. A more
recent study (Masuda et al., 2009) showed 545 E. coli membrane
proteins could be identified by increasing solubility of the mem-
brane proteins, as well as enhancing digestion efficiency using
phase-transfer surfactant, such as sodium deoxycholate (SDC), fol-
lowed by liquid chromatography–MS/MS analysis. Although the
non-gel-based approaches including combinatorial assays with n-
dimensional chromatography and MS for analysis of membrane
proteome show clear superiority over gel-based methods, the 2-DE
method has more benefits in some cases. The 2-DE method is read-
ily accepted and accessible for proteomic researchers. Also, since
the method allows itself to either absolute or relative quantification
of intact protein species using several complementary approaches,
the proteome information can be readily utilized to find potential
candidates for applying anchoring motifs as illustrated below.
In the biotechnology industry, the components of these outer
membrane proteins, such as E. coli FadL (Lee et al., 2004), LamB
(Sousa et al., 1998), OmpA (Lee et al., 2003b), OmpC (Xu and Lee,
1999; Choi et al., 2005) and TraT (Chang et al., 1999), M. magneticum
AMB-1 Mms13 (Yoshino and Matsunaga, 2006), and Pseudomonase
aeruginosa OprF (Lee et al., 2005a), have been widely used as
anchoring motifs for the display of recombinant proteins on the cell
surface of microorganisms. Microbial cell surface display has a wide
range of biotechnological and industrial applications, including
345
live vaccines, bioadsorbents, biosensors, whole-cell biocatalysts for
bioconversion, antibody production, screening of peptide libraries,
and mutation detection (Lee et al., 2003b). The choice of anchor-
ing motifs is important, since the wrong motif could result in the
destabilization of cell envelope integrity and growth defects. In
addition, different anchoring motifs often result in different display
efficiencies. Researchers can then use proteomic profiles of mem-
brane proteins to identify proteins that fit specific requirements,
such as an efficient signal peptide or transport signal to that allows
a premature fusion protein to be transported across the inner mem-
brane and a strong anchoring structure that keeps fusion proteins
on the cell surface without detachment.
The global protease protection assay (Wu et al., 2003) is a non-
gel-based proteomic approach for the identification of exposed
cell-surface peptides to correctly display a target on the outside of
cells. This assay can analyze protein topology and relative localiza-
tion by differentially digesting the exposed and protected domains
of membrane proteins using a combination of high pH and non-
specific proteolytic digestion as shown in Fig. 2. High pH disrupts
sealed membrane structures without denaturing the lipid bilayer
or extracting the integral membrane proteins, while proteinase K
cleaves the exposed hydrophilic domains of membrane proteins.
This approach permits the characterization of the relative cellu-
lar localizations of soluble proteins as well as the orientation of
membrane proteins (Blobel and Sabatini, 1970; Sabatini and Blobel,
1970). In addition, if this approach is combined with isotope-coded
affinity tag (ICAT) or iTRAG labeling methods, it may allow to quan-
tification of differences between samples. Therefore, this method
concurrently provides valuable data with regard to several aspects
of membrane proteins: (i) identification, (ii) quantification, (iii)
post-translational modifications (PTMs), and (iv) topology in the
membranes from biological samples. The relative sidedness of a
membrane protein is reflective of its cellular localization. The data
produced by this proteomic method has the potential to improve
the expression of membrane-bound proteins in biotechnological
applications.
A similar approach involves the identification of inner mem-
brane proteins that could be used as anchoring motifs to display
a target on the inner membrane. Interestingly, Majander et al.
(2005b) demonstrated the simultaneous display of three foreign
peptides in the FliD capping and FliC filament proteins of the E. coli
flagellum. This multi-hybrid display system has biotechnological
applications, such as the simultaneous delivery of several effec-
tor molecules or the introduction of sequential enzyme reactions
on the cell surface. Alternatively, more than two strains that dis-
play each different target can be co-cultivated for application to
a diverse range of fields. Thus the target display can be newly
applied or further improved by proteomic techniques for the iden-
tification and characterization of anchoring motifs that are robustly
integrated into the cell membrane. More importantly, both the
anchoring motifs and promoters described above can be identified
from proteomic studies, which will result in the increased improve-
ment of techniques for protein display in the near future.
2.6. Identification of fusion motifs from extracellular proteome
for excretory protein production
Bacterial excretory proteins are known to perform several
important functions, such as nutrient uptake, cell-to-cell communi-
cation, detoxification of harmful chemicals, and killing of potential
competitors (Tjalsma et al., 2000). Protein excretion occurs pri-
marily in Gram-positive bacteria, such as Bacillus subtilis, and in
only a few Gram-negative species, such as Pseudomonas species.
The increased availability of complete genome sequences allows
predictions to be made with regard to the composition of the
bacterial machinery for protein secretion as well as the extracellu-
346 M.-J. Han et al. / Journal of Biotechnology 145 (2010) 341–349

Fig. 2. Strategy for the identification of exposed cell-surface peptides to display a target on the outside of cells. A membrane protein is arbitrarily depicted with transmembrane
domains and soluble amino acid residues on both sides of the lipid bilayer. Amino acid residues are labeled with colored lines (outer exposed peptides on the membrane,
red; inner exposed peptides on the membrane, green; and transmembrane domains, black). In gel-based analysis, isolated membrane proteins are separated on gel matrix,
digested by a protease, such as trypsin and Lys-C/trypsin, and identified by mass spectrometry. In non-gel-based analysis, isolated membrane proteins are directly digested
by a protease and identified by coupled mass spectrometry; this procedure is often called shotgun proteomic analysis. Various peptides, including inner and outer exposed
peptides and transmembrane peptides, have been identified by both methods. Only outer exposed cell-surface peptides, however, can be identified by protease protection
analysis. Finally, the acquired peptides were analyzed to determine the orientation of the inner-exposed, outer-exposed, and trans-peptides using computational tools to
compare the predictive data. These results can be used for various biotechnological applications that involve the display of recombinant proteins on the cell surface.

lar complement of bacterial proteomes. The power of proteomics
was successfully employed to evaluate genome-based models of
these so-called secretomes. Many recent proteomic studies have
focused on the analysis of extracellular proteins from bacteria
that are commonly used in the biotechnology industry, such as
E. coli (Nandakumar et al., 2006; Xia et al., 2008), Bacillus sp.
(Tjalsma et al., 2000, 2004; Hirose et al., 2000; Voigt et al., 2006,
2009), and Aspergillus sp. (Medina et al., 2004; Oda et al., 2006)
and from pathogens, such as Helicobacter pylori (Bumann et al.,
2002), Edwardsiella tarda (Tan et al., 2002), Staphylococcus aureus
(Ziebandt et al., 2001; Jones et al., 2008), and Streptococcus mutans
(Len et al., 2003). The secretory proteome of pathogenic and non-
pathogenic strains may provide information on physiology of these
bacteria that would be applicable to the study of both pathogens
and strains used for the production of recombinant proteins.
In particular, one of the most salient features of Gram-positive
bacteria, such as B. subtilis and related bacilli, is their natural
capacity to secrete a variety of proteins, frequently at high concen-
trations, into their environment. This fact led to the commercial
exploitation of bacilli as major cell factories for secreted enzymes.
Progress in this field has been well-illustrated by the proteomic
analysis of protein secretion by the Gram-positive bacterium B.
subtilis, for which approximately 90 extracellular proteins were
identified (Tjalsma et al., 2004).
On the other hand, laboratory strains of E. coli can secrete
a trace amount of proteins beyond the inner and outer mem-
branes under normal culture conditions, as recently demonstrated
by genetic and proteomic tools (Nandakumar et al., 2006; Xia
et al., 2008; McBroom and Kuehn, 2007). Most of the periplas-
mic and outer membrane proteins can actually be released into
the culture medium in flask cultures (Nandakumar et al., 2006)
or in fed-batch, high cell density cultivation (Xia et al., 2008). E.
coli B strains are capable of releasing greater amounts of proteins
than the K-12 strains (Xia et al., 2008). Vesicle release by both
pathogenic and non-pathogenic Gram-negative bacteria is a ubiq-
uitous process that occurs over the course of normal growth. The
vesicle is composed of outer membrane and periplasmic materi-
als, which are released from the bacterial surface without the loss
of membrane integrity (McBroom and Kuehn, 2007). In addition,
a number of these proteins have been reported as being growth
phase-dependent (Xia et al., 2008), being significantly intensified
during the stationary phase (Tjalsma et al., 2004; Voigt et al., 2006)
and strictly regulated by environmental signals or under quorum-
sensing control. These observations suggest that the vesiculation
process can act to selectively eliminate unwanted material for
alleviating envelope stress, in which material that stresses the
envelope can be packaged as cargo into these vesicles and exported
out of the cell.
 M.-J. Han et al. / Journal of Biotechnology 145 (2010) 341–349
More importantly, some research groups have recognized the
importance of the excretory proteins of a variety of E. coli strains
and have identified a few major abundant proteins by both conven-
tional biochemical and proteomic techniques (Jeong and Lee, 2002;
Zhang et al., 2006). These proteins could potentially be used as
excretion fusion partners for the extracellular production of recom-
binant proteins. Excretory protein production has several attractive
benefits, including easy protein purification and increased chance
of correct folding. For example, an extracellular protein can be
used as a fusion partner to secrete a target protein into the culture
medium for simplified protein purification in downstream pro-
cesses. OmpF, which is a highly abundant extracellular protein in E.
coli BL21 (DE3), was successfully used for the secretion of recom-
binant human ␤-endorphin into culture medium (Jeong and Lee,
2002). Similarly, the excreted protein YebF, which was identified
in the culture supernatant of E. coli HB101, was used as a fusion
partner for the extracellular production of recombinant proteins
(Zhang et al., 2006). Another study demonstrated that recombi-
nant proteins that were fused to the 5 untranslated region of FliC
were secreted into the medium, via a modified flagellar type III
secretion apparatus (Majander et al., 2005a). In the extracellular
proteome of E. coli BL21 (DE3), Qian et al. (2008) recently identi-
fied twelve proteins that could be used as potential fusion partners
for the excretory production of recombinant proteins. The most
efficient excreting fusion partner, OsmY, has been used to excrete
heterologous proteins, such as E. coli alkaline phosphatase, B. sub-
tilis ␣-amylase, and human leptin, into the medium. Therefore,
extracellular proteomic data are very important to understanding
the phenotypic characteristics of the cell and can be used to develop
a system for the high-level extracellular production of recombinant
proteins from bacteria.
3. An integrated approach by proteomics combined with
more than one of the multi-omic techniques
Multi-omics technologies can be used for the large-scale anal-
ysis of biological systems at transcript, protein, metabolite, and
phenotype levels, which results in a wealth of information that is
useful in discovery-based science. Ideally, the information obtained
from each of these technologies will be integrated in order to
establish an in-depth understanding of the relationship between
genotype and any particular phenotype. There have been a few
reports that have demonstrated the successful engineering of
strains to enhance recombinant protein production based upon the
understanding of cellular physiology and metabolism using multi-
omics analyses that combined data from the proteome with data
from the transcriptome, metabolome, and/or fluxome.
An integrated analysis of transcriptomic and proteomic profiles
was carried out for E. coli W3110 and its l-threonine-overproducing
mutant strain (Lee et al., 2003a), in which the expression of
genes involved in glyoxylate shunt, the tricarboxylic acid (TCA)
cycle, and amino acid biosynthesis were shown to be significantly
up-regulated, whereas the ribosomal protein genes were down-
regulated. In addition, mutations in the thrA and ilvA genes involved
in biosynthetic pathways of amino acids were suggested to be
important for the overproduction of l-threonine. This combined
analysis provided valuable information with respect to the regu-
latory mechanism of l-threonine production and the physiological
changes in the mutant strain.
Another combined study involving proteome, transcriptome,
and mathematical models was used to engineer a specific E. coli
strain (Lee and Lee, 2005). An E. coli mutant strain, which was
obtained by random mutagenesis and which secreted 4-fold more
active ␣-hemolysin (HlyA) than its parent strain, was characterized
using both high-density microarrays for mRNA profiling and 2-D
347
gels for protein profiling. In particular, the relative mRNA and pro-
tein expression levels of tRNA-synthetases, including AsnS, Asps,
LysS, PheT, and TrpS, were decreased in the mutant strain compared
to the parent strain. This combined examination of the mRNA and
protein expression profiles demonstrated that the down-regulation
of the tRNA-synthetases in the mutant reduced the general transla-
tion rate and more specifically lowered the rate of HlyA synthesis.
Thus, the improved secretion of active ␣-hemolysin at low syn-
thesis rate can be attributed to a balance between translation and
secretion. The use of rare codons in the hlyA gene has been shown
to reduce its rate of translation, since the number of available
aminoacyl-tRNAs is limited. A variant of the hlyA gene, in which
five bases are altered but the amino acid sequence remained the
same, was designed using a mathematical model of prokaryotic
translation. As a result, the rate of translation could be artificially
slowed down in this variant, which led to further improvements in
the secretory production of active ␣-hemolysin.
Successful examples of integrating ‘-omics’ studies and compu-
tational analysis for the development of improved strains continue
to be generated (Lee et al., 2005b). This multi-omic systems
approach for the development of optimized strains will become
more and more popular, since it can identify potential new targets
for various biotechnical applications in a more systematic manner.
4. Conclusion
Proteomics is one of the most important tools in biological
research and in various biotechnological applications. Since pro-
teomics alone is not sufficient to understand cellular physiology
and regulatory mechanisms as a whole, combined analysis or multi-
omic approaches will become increasingly important. This type of
integrated analysis will lead to an improved understanding of cel-
lular physiology and metabolism at the systems level and will pave
the way towards more efficient metabolic engineering. There have
been difficulties, however, with attempts to completely integrate
the data generated by these multi-omic techniques into a complex
biological process, because a large amount of false positives and/or
true negatives is generated as a result of some technical limitations
and because there are frequent inconsistencies between mRNA
and protein levels under the same conditions. For these reasons,
more highly sensitive and accurate instruments, integrated tools,
and advanced techniques will need to be continuously developed,
which will allow complete biological systems to be monitored at
the molecular levels and, in turn, will allow the creation of custom-
made producing strains that are suitable for a variety of industrial
processes.
Acknowledgments
This work was supported by the Converging Research Center
Program through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology (No.
2009-0093652).
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