ANALYTICAL SCIENCES JANUARY 2009, VOL.

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2009 The Japan Society for Analytical Chemistry

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Reviews

Molecular Display Technology Using YeastArming Technology

Seiji SHIBASAKI,* Hatsuo MAEDA,* and Mitsuyoshi UEDA**
*Laboratory of Bioanalytical Chemistry, Department of Pharmacy, School of Pharmacy,
Hyogo University of Health Sciences, Minatojima, Chuo, Kobe 6508530, Japan
**Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School of
Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo, Kyoto 6068502, Japan

A technology to display proteins or peptides on living organisms has been developed over the last two decades. So-called
Molecular display (Arming) technology or Cell surface engineering has been one of the important tools for analyzing
and understanding protein function, or for screening of novel clones from libraries. In addition, it endows cells with
novel abilities that cannot be added by conventional genetic recombination. In particular, the yeast Saccharomyces
cerevisiae has a lot of advantages in molecular display technology. Normal yeast cells can be transformed into a wide
variety of arming yeasts that have catalytic functions, affinity binding to valuable ligands, bioremediation properties,
bio-monitoring properties, etc. This review describes the background, applications, and representative achievements of
molecular display technology of yeast.
(Received October 23, 2008; Accepted November 17, 2008; Published January 10, 2009)

1 Introduction
41
11 Overview of molecular display
12 Architecture of the scaffold in the molecular
display of yeast
13 Principle of the molecular display of yeast
2 Immobilization of Functional Proteins or
Peptides on Yeast Cell Surface
43
21 Display of enzymes as whole-cell biocatalysts
211 Amylolytic enzymes
212 Cellulolytic enzymes
213 Lipase
22 Display of binding proteins as adsorbents in
environmental processes

1 Introduction
11 Overview of molecular display
Cells and viruses have a large number of molecules inside and
maintain their physiological conditions by certain protein and
lipid complexes, such as the cell membrane, cell wall or coat
proteins. These are not only boundaries between the cytosol and
the extracellular environment, but also important factors for
communication between the inside and outside of a cell, for
exchange of molecules, for recognition of proteins or other
organisms, and so on. For instance, antigens are recognized by
receptors displayed on T-cells or B-cells, and CD4 or CD8
molecules displayed on T-cells are required to bind with MHC
molecules for effective immune responses.1 Moreover, many
kinds of receptors coupled with enzymes are located on the
surface of a wide variety of cells to receive proteins, peptides, or
To whom correspondence should be addressed.
E-mail: seiji@huhs.ac.jp

23 Display of antibodies and related molecules

for diagnostics and therapeutics
3 Sensing and Monitoring
31 Exploration of the cell wall by a visible
reporter
32 Response to environmental changes
33 Monitoring of the production of bioactive
compounds
34 Other types of molecular display for sensing
molecular interactions
4 Combinatorial Libraries
5 Summary
6 References

45

46
47
48

other compounds as signal molecules, and transduction of

information into the cytosol or nucleus occurs after ligandbinding.2 Thus, natively displayed molecules on the surface play
important roles in various biological or physiological phenomena.
Genetic engineering was established in the 1970s, and
molecular display technology has emerged since the 1980s.
Phage surface display was the first technology used. Smith3
showed that a foreign protein could be inserted into filamentous
phage protein III, and its fusion protein was displayed on the
virion surface. His colleagues and others have developed it into
a methodology for screening peptide ligands or combinatorial
protein clones.46
However, in that screening system, a
complicated process using infection of Escherichia coli cells
with phage displaying positive clones is needed for recovery.
Furthermore, formations of larger-sized peptides or proteins
with coat proteins of phage are not suitable for surface display.
However, bacterial cells provide a more suitable surface for
displaying large sizes and numbers of proteins.7,8
E. coli was initially used as a Gram-negative bacterium in
molecular display technology.912 For example, the outer

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ANALYTICAL SCIENCES JANUARY 2009, VOL. 25

Fig. 1 Architecture of the yeast cell wall. SMP, glucanase-extractable

surface-layer mannoprotein; PP, SDS-extractable periplasmic protein.

membrane proteins OmpA9 or Intimin EaeA;11 fimbriae;12 or the

flagellar protein flagellin13 function well as anchors of foreign
proteins on the cell surface. On the other hand, Gram-positive
bacteria have also been studied as hosts of molecular display.
Sthl et al. have discovered applications for molecular display
with Staphylococcus,14,15 and there is interest in using
Lactobacillus and Lactococcus to feasibly anchor foreign
proteins; e.g., Bacillus subtilis subsp. chungkookjang PgsA16 or
Streptococcus pyogenes M6 protein can be used for displaying
target proteins,17 in addition to a proteinase existing on the
foreign cells surface.18 Although diverse microorganisms have
been investigated for displaying foreign proteins, the expression
of eukaryotic proteins is sometimes difficult in the bacteria
mentioned above.
The yeast Saccharomyces cerevisiae is very useful as a host
cell in genetic engineering because it folds and glycosylates
heterologous eukaryotic proteins. S. cerevisiae also has the
advantage of high density cultivation in inexpensive medium. In
addition, the yeast has a potential to display other eukaryotic
proteins, and can display different kinds of protein on the same
cell surface, namely co-display. Several auxotrophic markers
can be used in genetic manipulation of yeast cell to display
proteins. Moreover, a flow cytometer is applicable for yeast
cells in the case of high-throughput screenings. Therefore,
molecular display using the yeast cell surface has many potential
benefits and practical applications.
In this review, we focus on the current development of molecular
display technology using yeast cells.1921 First, we describe the
structure of the yeast cell surface and the principles of molecular
display technology, including representative applications. Next,
we describe important achievements in both sensing and protein
library screening, and then discuss future directions.
12 Architecture of the scaffold in the molecular display of
yeast
The yeast S. cerevisiae cell is surrounded by a hard cell wall
(Fig. 1) that consists of b-linked glucans and mannoproteins,22
and exists outside of the plasma membrane. The cell wall
consists of an internal skeletal layer of glucan, composed of
b-1,3- and b-1,6-linked glucose,23,24 and a fibrillar or brush-like
outer layer composed predominantly of mannoproteins.25 There
are two types of mannoprotein in the thick cell wall.26 One is
loosely bound to the cell wall with non-covalent bonds, and is
extractable with sodium dodecylsulfate (SDS). Approximately
60 proteins are released from the cell wall by solubilization with
hot SDS.27 The other type is extractable by digestion of the cell

Fig. 2 Schematic illustration of GPI anchor. An anchored protein is

released with cleaving by phosphatidyl inositol-specific phospholipase
C (PI-PLC).

wall with b-1,3- or b-1,6-glucanase.28

Many glucanase-extractable mannoproteins are present on the
yeast surface; for instance, agglutinin (Aga1 and Aga2), Flo1,
Sed1, Cwp1, and Tip1 have glycosylphosphatidylinositol (GPI)
anchors composed of ethanolamine phosphate-(6)mannose
(a1,6)mannose(a1,4)glucosamine(a1,6)-inositol phospholipids
(Fig. 2).2933 GPI anchors have been found on many eukaryotic
plasma membrane proteins and play important roles for cellsurface proteins, and in addition are essential for the viability of
yeasts. These glucophospholipids are covalently linked to the
C-termini of proteins, and their primary function is to allow
stable association of proteins with the membrane.
Localization of GPI-anchored proteins on the cell surface
takes place through the secretory pathway, exocytosis. Protein
secretion in S. cerevisiae comprises transfer through various
membrane-enclosed compartments. Secreted proteins are first
translocated into the lumen of the endoplasmic reticulum (ER)
to the Golgi apparatus and from there to the plasma membrane
in membrane-enclosed vesicles.34,35 The fusion of the Golgiderived secretory vesicles with the plasma membrane releases
the secreted proteins outside the cytosol. Post-translational
proteolytic modification of precursors of secretory peptides
takes place in the late secretory pathway (in the trans cisternae
of the Golgi apparatus and secretory vesicles). In S. cerevisiae,
the Kex2 endopeptidase is situated in the trans cisternae of the
Golgi apparatus to eliminate the proregion of precursors, such as
the a-factor pheromone used in mating. a-Agglutinin is thought
to be further transported to the exterior of the plasma membrane
by secretory vesicles in a GPI-anchored form, and then released
from the plasma membrane by phosphatidylinositol-specific
phospholipase C (PI-PLC) and transferred to the cell wall. The
anchorage of a-agglutinin in the outermost surface of the cell
wall is accomplished by the addition of b-1,6-glucan to the GPI
anchor remnant, in a manner dependent on the prior addition of
a GPI anchor.3638
A number of examples of molecular display using the
representative GPI-anchored proteins, a-agglutinin and flocculin,
are described in a later section.
13 Principle of the molecular display of yeast
Flo1, Cwps, and a- and a-agglutinins have been utilized for
the display of proteins on a yeast cell surface. Flo1 is a lectinlike cell-wall protein and plays a major role in flocculation.39,40

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It is composed of the following characteristic domains: secretion

signal, flocculation functional domain, GPI-attachment signal,
and membrane-anchoring domain. There are two types of
molecular display technologies using Flo1.21 One is the GPI
system using the C-terminal region of Flo1 which contains a
GPI-attachment signal, and the C-terminus of the target protein
fused to that of Flo1.41 Another system is based on the adhesive
ability of the flocculation functional domain of Flo1.42 In that
case, the N-terminus of the target protein is fused to the
flocculation functional domain, and as a result, the target protein
is displayed on the cell surface with its C-terminus free, in
contrast to the GPI system. Although Flo1-based systems are
useful in some cases, we mainly discuss molecular display
systems using a more attractive scaffold, agglutinin, in this review.
On the cell surface of S. cerevisiae, there is one of two
different types of agglutinins during mating. Mating type a and
a cells express a-agglutinin and a-agglutinin, respectively.
a-Agglutinin in a-type cells is encoded by AGa1 and interacts
with the binding subunit of a-agglutinin of a-type cells.
a-Agglutinin has a core subunit encoded by AGA1 and a binding
subunit encoded by AGA2.33,43 Both a-agglutinin and the core
subunit of a-agglutinin consist of a secretion-signal region, a
functional region, a support region, and a putative GPI anchorattachment signal. To display foreign proteins on the cell wall,
genetic information of each agglutinin is utilized. The cell wallanchoring region of a-agglutinin is combined with the secretion
signal sequence by genetic engineering (Fig. 3A). Fusion to the
C-terminal half of a-agglutinin is used to anchor foreign proteins
on the yeast cell surface (Fig. 3B). In the case of a-agglutinin,
secretion type protein Aga2, the binding subunit linked by a
disulfide bond to the core protein Aga1, is used.44,45 Eventually,
heterologous proteins are covalently linked with the glucan-layer.

2 Immobilization of Functional Proteins or

Peptides on Yeast Cell Surface
21 Display of enzymes as whole-cell biocatalysts
The display of enzymes is one of the most attractive
applications of molecular display technology in yeast. Although
the yeast S. cerevisiae has long been utilized in fermentation for
the production of food, pharmaceuticals, bioactive compounds,
alcohol, etc., arming the yeast cell surface with foreign proteins
enhances their abilities and potentials as whole-cell biocatalysts.
211 Amylolytic enzymes
Yeasts play a critical role in the commercial production of
ethanol from starchy materials. In the conventional procedure,
crude material is prepared at 140 180C prior to amylolysis.
To conserve energy in this process, pioneering studies used the
enzymatic degradation of raw starchy materials by amylolytic
enzymes.46,47 Ashikari et al. examined the construction of yeast
cells secreting glucoamylase, an exo-type amylolytic enzyme,
derived from Rhizopus oryzae for the fermentation of raw starch
without cooking.4850 Amylolytic enzyme-displaying yeast cells
may be able to saccharify starch on their surfaces and assimilate
the released glucose to grow and ferment. Therefore, the strain
displaying R. oryzae glucoamylase was created by a fusion
protein with a-agglutinin under the control of the GAPDH
promoter.51,52 The strains were demonstrated to have the
displayed glucoamylase activity without secretion of the active
enzyme during cultivation in medium containing 2% glucose.
When the strains were cultivated aerobically with 1% soluble
starch as the sole carbon source, cells proliferated to the same
level as that of yeasts cultured on 1% glucose. However, no
growth was observed with the control strains. These results

Fig. 3 Principle of cell surface display. A, Genetic construction of

target-a-agglutinin-fusion protein; B, confocal laser scanning
micrograph of EGFP-displayed cell.

suggested that glucoamylase-displaying cells sufficiently

catalyzed the hydrolysis of starch.
Next, the ethanol production of the glucoamylase-displaying
strain was examined, and found to show high productivity from
soluble starch.
However, an insoluble starch fraction
accumulates during the fed-batch process. To improve ethanol
productivity from starchy materials, an endo-type amylolytic
enzyme a-amylase [EC 3.2.1.1] was added to this system. For
the co-display of glucoamylase and Bacillus stearothermophilus
a-amylase53 on the same cell surface, both glucoamylase/
a-agglutinin- and a-amylase/a-agglutinin-encoding genes were
integrated into the yeast chromosomes.54 The constructed strain
that harbored co-displayed glucoamylase and a-amylase grew
faster than the glucoamylase-displaying cells. In addition, the
co-displaying strain effectively improved ethanol productivity
from starchy materials.
212 Cellulolytic enzymes
The desire to produce ethanol with non-fossil resources has

44
increased year by year. The use of cellulosic biomass is more
desirable than that of starchy materials, because it creates no
conflict with the high social demand for food. Cellulosic biomass,
such as agricultural and forestry residues, waste paper and waste
lumber, could be used as an inexpensive and abundantly
available source of sugar for ethanol production. However, S.
cerevisiae is unable to utilize cellulosic materials, which must
be subjected to saccharification to glucose before fermentation.
To solve this problem, a variety of cellulolytic enzymes, for
example carboxymethylcellulase (CM-cellulase) and b-glucosidase
(BGL1) from Aspergillus aculeatus, have been co-displayed on
the yeast cell surface. This co-displaying strain was grown in a
medium supplemented with cellooligosaccharides as the sole
carbon source.55 In addition, endoglucanase II (EGII) from
Trichoderma reesei was also co-displayed with BGL1 on the
yeast cell surface, which was then grow in a medium containing
b-glucan as the sole carbon source. It directly fermented 45 g
b-glucan/l to produce 16.5 g ethanol/l within about 50 h.56
The molecular display of b-glucosidase has also been
developed in the production of bioactive compounds. Kaya
et al. constructed a strain displaying b-glucosidase cloned from
A. oryzae on the cell surface, and examined isoflavone
aglycones production from isoflavone glycosides by the strain.57
Isoflavone aglycones are hydrolysates of isoflavone glycosides
by b-glucosidase, and are highly effective in the prevention of
several diseases. First of all, putative genetic sequences of
b-glucosidases were cloned from koji mold A. oryzae, because it
was know that soybean paste fermented by koji molds includes
plenty of isoflavone. BGL1, BGL3, and BGL5-encoding genes
were isolated and displayed on the cell surface of Sake yeast
under control of the SED800 promoter. The strain displaying
BGL1 exhibited the highest activity, and also had the broadest
substrate specificity to isoflavone glycosides.
213 Lipase
The lipase of R. oryzae (ROL) has relatively high activity, and
features such as its stereoselectivity and maturation process have
been well studied. In addition, ROL is very similar to other
fungal lipases in its structure. The molecular display of
Humicola lipase and Fusarium cutinase exhibit low-level
activity against p-nitrophenyl butyrate, but no activity against
olive-oil emulsion.58 The lack of activity is thought to be due to
a lack of lipid binding to the enzymes, since the active site is
situated near the C-terminal region in Rhizopus lipase. Washida
et al. showed the effectiveness of introducing spacer-amino
acids, a Ser/Gly repeat sequence, into the boundary of ROL and
a-agglutinin.59 The linker peptides enhanced enzyme activity,
with a tendency for the extent of triolein hydrolysis to increase
as the peptide becomes longer. To further improve the activity
of enzymes, such as ROL, whose active site is near the C-terminus,
Matsumoto et al. constructed strains displaying ROL based on
Flo1.60 In this system, the activity of the displayed lipase
reaches a much higher level than the above systems, indicating
its effectiveness in displaying proteins with active sites located
near their C-termini. In recent years, Candida antarctica lipase
B (CALB) has been displayed based on both a-agglutinin- and
Flo1-systems to develop more of a practical whole-cell catalyst
for industrial use.61,62 The thermal stability of CALB displayed
on a yeast surface was higher than that of ROL.
22 Display of binding proteins as adsorbents in environmental
processes
There are two ways to recover heavy-metal ions using
microorganisms. One is binding the metal ions to cell-surface
components; the other is gradual uptake and intracellular
sequestration. Although the recovery of intracellularly accumulated

ANALYTICAL SCIENCES JANUARY 2009, VOL. 25

metal ions requires disruptive treatment of cells, metal ions
adsorbed on the cell surfaces can be recovered with simple
chemical processes, such as separation by EDTA, etc.
Adsorption on the cell surface by metal ion-binding componentdisplaying cells is thought to be useful for the bioremediation of
heavy-metal pollutants because the yeast cells can be easily
cultivated and recycled in the recovery process of heavy metal
ions. Practical use of cells as adsorbents of heavy metal ions
requires examining resistance or sensitivity to those ions. A
histidine oligopeptide (Hexa-His) with the ability to chelate
divalent heavy metal ions (Cu2+, Ni2+, etc.) has been displayed
on the yeast cell surface to enhance adsorption.63 This strain
adsorbs three to eight times more copper ions than that of the
parent strain; in addition, it also has more resistance to copper
(4 mM) than the parent strain (below 1 mM, at pH 7.8). It is
possible to recover about a half of the copper ions adsorbed by
whole cells with EDTA treatment without disintegrating the
cells. To develop this strain for practical use as a bioadsorbent,
a novel separation system was examined. In general, the
cultivated medium is centrifuged for the separation of cells;
however, an expensive device is required for that process.
Therefore, Kuroda et al.64 endowed the strain with the ability to
self-aggregate in response to binding and accumulation of
copper ions by overexpression of GTS165,66 under a copper ioninducible genetic element, the CUP1 promoter.67 The copper
ion-induced aggregation did not interfere with the copper ionadsorbing function of the Hexa-His-displaying yeast, suggesting
that the strain is endowed with the ability to be removed easily
from treated water. Furthermore, yeast metallothionein (YMT)
was tandemly fused and displayed on the surface.68 The
adsorption and recovery of Cd2+ on the cell surface was
increasingly enhanced with increasing numbers of tandem repeats.
Interestingly, cells displaying eight YMT repeats were able to be
grown in the presence of levels of Cd2+ that were toxic to others.
To evaluate the ability of chemical compounds acting as
endocrine disruptors to bind to steroid hormone receptors,
various in vivo and in vitro assay systems have been developed
and used to determine estrogenicity.6971 The molecular display
of the ligand-binding domain of the rat estrogen receptor
(ERLBD) was examined to analyze and entrap endocrine
disruptors. The ligand-binding ability and the number of
molecules displayed on each cell were evaluated using the
fluorescent 17b-estradiol (17-FE) binding assay system.72 This
indicated that cells displaying ERLBD on the surface were
saturated at 9.5 104 molecules/cell. The number of 17-FE
molecules entrapped on the surface suggested that a system
based on the molecular display of the yeast is valuable not only
for screening hormone-like compounds through competitive
experiments, but also in removing them from the environment.
23 Display of antibodies and related molecules for diagnostics
and therapeutics
As a binding protein module for immunoglobulin G (IgG), the
Z domain derived from the B domain of Staphylococcus aureus
Protein A (SPA) has been developed by Nilsson et al.73 The Z
domain has a three-helix bundle structure, high solubility, and
high stability. It binds to the Fc fragment of human or rabbit
IgG, and is commercially available as the ZZ domain repeated
with the Z domain for the purification of IgG from blood
samples. The ZZ domain has also been used as an affinity tag to
purify recombinant proteins and in immunoassays. Nakamura
et al. constructed cells displaying the ZZ domain on their
surface, and examined their effectiveness as immunoadsorbents
in an enzyme-linked immunosorbent assay (ELISA) and
sandwich ELISA.74 They revealed that the ZZ domain-

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displaying cell detects IgG and antigens down to a concentration
of 1 10 ng/ml. The detection range is broad and can be varied
by adjusting the cell concentration and the duration of the
reaction with the enzyme substrate. To further improve the
amount of displaying ZZ domain on the surface, various
promoters for expression of the secretion signal sequence-ZZ
domain/a-agglutinin-encoding gene, yeast host strain, and
cultivation conditions (duration and pH) have been examined.75,76
The molecular display of the ZZ domain was successfully
enhanced under the control of the GAL1 promoter using the
BY4742 strain as the host in the optimized cultivation
conditions. In addition, this improved ZZ domain displaying
strain was use to develop a novel synergistic system for the
production and recovery of secreted recombinant proteins. The
Fc fragment was fused to the enhanced green fluorescent protein
(EGFP) or lipase ROL and secreted into the medium. At the
same time, the ZZ domain-displaying cells were cultivated in
the same flask, and the Fc fusion proteins secreted by the other
strain were fixed with the ZZ domain of the cell surface. After
the cells were treated with an acidic solution, the Fc-fusion
protein was found in the eluted fraction. Thus, it was revealed
that the Fc fusion protein could be precisely bound to the ZZ
domain displayed on the cell surface, and it could be separated
and recovered by cultivation only. This synergistic production
and recovery system provides a convenient and lower cost
production system of secreted recombinant proteins for
biotechnological or pharmaceutical industries.
Similar to the IgG binding Z domain, antibodies themselves
have been successfully displayed on the yeast cell surface as
examples of molecular display for hetero-oligomeric proteins.
This is made possible by the eukaryotic characteristics of yeast,
namely post-translational modification, efficient disulfide
isomerization, and vesicle transport the same as in mammalian
cells. As a model, a hydrolytic antibody 6D977 was displayed on
the yeast cell surface.78,79 A hetero-oligomeric Fab fragment of
6D9 hydrolyzed a non-bioactive chloramphenicol monoester
derivative to produce choloramphenicol in an active form. To
display 6D9-Fab containing the heavy chain (Fd fragment) and
light chain (Lc fragment), the Fd and Lc fragments were
expressed independently (Fig. 4). While one fragment was
anchored by a-agglutinin, the other was expressed using an
identical promoter and secretion signal sequence. The Fab1
displayed by Lc-a-agglutinin and Fd was superior to Fab2
containing Fd-a-agglutinin and Lc in a binding assay using
transition-state analog 3 (TSA3). It was also confirmed that
these Fab fragments formed in the transport vesicle, and this
suggests that hetero-oligomeric proteins can be displayed on the
yeast cell surface by the anchor of a-agglutinin.

3 Sensing and Monitoring

Non-invasive sensing or monitoring techniques are an important
objective for bioprocesses or studies of molecular and cell
biology. As described above, molecular display technology has
shown that yeast surfaces provide us with spaces to immobilize
desired proteins in their active form. A reporter protein that is
able to emit fluorescence would provide us with intra- or
extracellular information if displayed on the yeast cell surface.
Although various kinds of fluorescent proteins are available, the
green fluorescent protein (GFP) from jellyfish Aequorea victoria
has been especially useful as a reporter in bacteria, fungi,
mammalian, or plant cells to analyze localization or functions of
target proteins.8082 A cell surface displayed fluorescent probe
emitting in response to any given analyte might be applied for

45

Fig. 4

Fab-display on the yeast cell surface.

sensing environmental changes inside or outside of living cells.

31 Exploration of the cell wall by a visible reporter
The first studies examined whether the display of fluorescent
proteins on yeast cell surfaces would succeed or not.83 The GFP
gene was fused with the gene encoding the C-terminal half of
a-agglutinin and the secretion signal sequence of the fungal
glucoamylase protein. This designed gene was integrated into
the chromosome of the S. cerevisiae MT8-1 strain with
homologous recombination. The display of GFP on the cell
surface was confirmed using microscopic analysis and by
measuring the fluorescence. Moreover, display of GFP was
further verified by immunofluorescence labeling with a
polyclonal antibody against GFP as the primary antibody and
rhodamine RedTM-X-conjugated goat anti-rabbit IgG as the
second antibody. Next, the enhanced GFP (EGFP), which has
35-fold more fluorescence than wild-type GFP,84 was displayed
on the surface by the same method, because EGFP absorbs 488
nm light emitted from an argon-ion laser, and is suitable for
analysis by confocal laser scanning microscopy (CLSM)
(Fig. 3B).85 Although cell surface EGFP was affected by changes
in pH during cultivation, this was solved by adding HEPES into
the medium and adjusting to pH 7.0 before starting cultivation.
Using this stabilized condition to measure fluorescence, EGFP
molecules displayed on the surface were quantitatively evaluated
to answer the question; How many target proteins can be
displayed on the yeast cell surface? CLSM image analysis was
based on extraction of fluorescence from cell surfaces, not
including autofluorescence from the cytosol, by processing all
serial sections taken by laser scanning microscopy. The strain
harboring a genome-integrated fusion gene encoding EGFP-aagglutinin has shown 104 molecules on the surface, while the
strain harboring a multi-copy type plasmid encoding the same
gene has shown 105 molecules on the surface.
32 Response to environmental changes
As GFP on the cell surface is displayed in its active form,
selection of promoters should endow yeast cells with the ability
to respond to changes in concentrations of molecules of interest
which induce expression with those specific promoters (Fig. 5).
The above constructed strain displaying GFP emits under the
control of the GAPDH promoter which functions when glucose
is present in the medium.
As a next step, another sensing element to respond to the
exhaustion of glucose was introduced.
The UPR-ICL
promoter86,87 from Candida tropicalis, which has been found to
be functional in S. cerevisiae, was selected to sense the
exhaustion of glucose, with one of the GFP variants, BFP (blue
fluorescent protein), used as the second reporter.88 BFP contains
three amino acid substitutions, so that the emission is shifted
from green to blue.89 Under the UPR-ICL promoter, genes
encoding the secretion signal sequence, BFP, and the 3-half of

46

Fig. 5 Overview of monitoring by the engineered yeast cells which

responds to environmental changes. XFP1 or XFP2 gene codes
different color fluorescent protein, and are induced by different
analytes via each promoter, reslpectively.

a-agglutinin which functions as a cell wall anchoring region

were placed in a vector for integration into the yeast genome.
Fluorescent and immunofluorescent microscopy demonstrated
the display of BFP in addition to GFP. The relationships
between the concentrations of intra- or extracellular glucose and
fluorescence were defined, and in addition, the switching of
GFP and BFP fluorescence was evaluated.88
In another study, two kinds of promoters derived from S.
cerevisiae, which are functional under nutrient limitation, were
selected. The PHO5 promoter is the upstream sequence of the
gene PHO590,91 for a secreted acid phosphatase. Transcription
of PHO5 is known to be regulated by the extracellular
concentration of inorganic phosphate, i.e., repressed in highphosphate medium and derepressed in a low-phosphate medium.
On the other hand, the MEP2 promoter is the upstream sequence
of the gene MEP2,92,93 which encodes an ammonium ion
transporter protein. Recent evidence has shown that there are
three MEP transporters, and they are required to retain NH4+
inside cells during growth on minimal nitrogen sources other
than NH4+. In the presence of a good nitrogen source (e.g.,
glutamine, asparagine, or ammonium ion), all three MEP genes
are repressed. On a poor nitrogen source, MEP2 expression is
much higher than MEP1 and MEP3 expression. PHO5 and
MEP2 promoters were used as elements to make cells to
respond to environmental changes. For information output by
the yeast cell surface, two kinds of reporters, ECFP (enhanced
cyan blue fluorescent protein) and EYFP (enhanced yellow
fluorescent protein), which are variants of GFP, were used.94
Thereafter, flow cytometric studies showed that the fluorescence
emission from ECFP or EYFP displayed on the yeast cell
surface reflected the intra- and extracellular concentrations of
phosphate or ammonium ions. The relationships between each
ion concentration inside and outside the cells was evaluated by
the change in the rate of fluorescence.
These systems will provide us with a sensing tool for
environmental changes which can output intra- and extracellular
information using suitable promoters in real-time and in a noninvasive manner.
33 Monitoring of the production of bioactive compounds
In the first application of GFP in bioprocess monitoring,
Poppenborg et al. reported the monitoring of a hisactophilinGFP fusion in bacterial fermentation.95 Practical products from
bacteria can now be monitored by fluorescence of GFP. For
instance, organophosphorous hydrolase (OPH), CAT, and human
interleukin-2 (hIL-2) were fused to GFP and expressed in
E. coli.96 In these cases, although active proteins were finally

ANALYTICAL SCIENCES JANUARY 2009, VOL. 25

produced, they had to be processed by proteolytic enzymes. For
the practical use of GFP as a reporter in bioprocesses, it seems
like that GFP should be expressed independent of target proteins
without fusion.
Molecular display of GFP described above was examined to
demonstrate the effectiveness in monitoring systems for
production of foreign protein in yeast cells. As a model, the
GAL1 promoter97 was selected to regulate gene expression. The
GAL1 gene is induced, at the level of transcription, 1000-fold by
growth with galactose. Molecular display of EGFP on the
surface was well-controlled by the GAL1 promoter, and two
kinds of proteins were selected to be produced intra- or
extracellularly, whose production is monitored by the
fluorescence of EGFP displayed on the cell surface. One is
b-galactosidase derived form E. coli,98 which was used as an the
example of an intracellular protein to be monitored. Another is
human interferon w (IFN-omega) as an extracellular protein.
IFN-w is secreted by virus-infected leukocytes as a major
component of human leukocyte interferon.99 These proteins
were produced under the control of the same promoter as that
for displaying EGFP on the surface.100 Both proteins to be
monitored and the reporter EGFP were produced under the
control of the GAL1 promoter. Protein production, the intraand extracellular concentrations of galactose which was the
carbon source, and fluorescence from EGFP displayed on the
cell surface were monitored and correlated up to 24 h in each
cultivation. These cells can be used for non-invasive monitoring
of protein production and components of media in bioprocesses.
34 Other types of molecular display for sensing molecular
interactions
A molecular display using novel reaction sites and interaction
sites has been developed which does not depend on the
a-agglutinin or Flo1 systems. This molecular display system
was created using the cytoplasmic face of a plasma membranetargeting system in yeast.101 The C-terminal transmembrane
domains (CTM) of Ras2p102,103 and Snc2p,104,105 which have
anchoring ability to the cytoplasmic face of the plasma
membrane, were examined. It was found that the CTM of
Snc2p targeted the ECFP-Z-domain fusion protein on the
cytoplasmic face of the plasma membrane more strongly than
that of Ras2p. To develop it for use as a detection system for
protein-protein interactions, the Fc fragment of IgG was
genetically fused with the EYFP and expressed in the cytoplasm
of the ECFP-Z-domain-anchored cells (Fig. 6). Microscopic
analysis showed that fluorescence resonance energy transfer
(FRET) between ECFP-Z-domain and EYFP-Fc occurred, and a
change in fluorescence was observed on the cytoplasmic face of
the plasma membrane. The detection of protein-protein
interactions at the cytoplasmic face of the plasma membrane
using FRET combined with a cytoplasmic molecular display
system provides a novel method for examining the molecular
interactions of cytoplasmic proteins, in addition to other types of
detection methods, such as the two-hybrid method in the
nuclei106 or the protein fragment complementation assay in
cytosol.107,108

4 Combinatorial Libraries
Combinatorial bioengineering is a powerful technique for
designing DNA libraries that rapidly translate functional
proteins or peptides by several molecular display technologies.
Newly synthesized proteins exhibiting desired properties are
directly selected among a highly diversified library, including

ANALYTICAL SCIENCES JANUARY 2009, VOL. 25

Fig. 6

47

Anchoring of proteins in the cytoplasmic face to observe protein-protein interactions.

combinations of the 20 amino acids. Molecular display

technology of yeast has contributed to the developmental of
combinatorial libraries. Several representative examples in
display technology are introduced below.
As a first example, a combinatorial protein library constructed
through displaying about 4 104 independent clones on yeast
cells was screened against selective pressure of n-nonane.109
The library was constructed by random priming recombination
(RPR) using mRNA as a template with random primers, and
then introduced into the yeast MT8-1 strain. As a result, only
one clone retained nonane tolerance after several passages of
cultivation in the presence of the solvent. This strain is the first
genetically constructed recombinant yeast strain able to tolerate
the organic solvent n-nonane, and may lead to a much wider
application of yeasts in industrial bioprocesses.
As a second example, combinatorial libraries of the lid domain
of lipase ROL were displayed on the yeast surface.110 Several
clones exhibiting unique substrate specificity were obtained by
halo assays and an assay using fluorescent substrates. Based on
computer modeling of these mutants, two mutants of ROLs with
a significant shift in substrate specificity were obtained.
Computer modeling of these mutants suggested that they form a
unique oxyanion hole. Therefore, molecular display technology
coupled with computational modeling can be used to create
novel proteins and examine them in combinatorial bioengineering.
As a third example, some trials for controlling of substrate
specificity of a peptidase were performed. First, Lc-WT, the
wild-type light chain of the antibody, and Lc-Triad, its double
mutant with E1D and T27aS designed for the construction of a
catalytic triad with the original Asp1, Ser27a, His93 residues,
were displayed on the surface of a protease-deficient yeast strain.111
Lc-Triad-displaying cells showed higher catalytic activity than
Lc-WT-displaying cells. The activity disappeared in the presence
of the serine protease inhibitor diisopropylfluorophosphate,
suggesting that the three amino acid residues functioned as a
catalytic triad responsible for the proteolytic activity in a similar
way to the anti-vasoactive intestinal peptide (VIP)112 antibody
light chain. A serine protease-like catalytic triad (Ser, His, and
Asp) is considered to be directly involved in the catalytic
mechanism of the anti-VIP antibody light chain, which
moderately catalyzes the hydrolysis of VIP. This result suggests
that the approach may be used for the creation of tailor-made
proteases beyond traditional limitations by immunization. Next,
the alteration of substrate specificity of a peptidase was
examined using yeast molecular display technology.113 There
are few reports on the modification of substrate specificity of
peptidases. Neurolysin [EC 3.4.24.16]114 is classified as a

metalloendopeptidase cleaving the bioactive peptide neurotensin,

and was rapidly modified by semirational mutageneis using
molecular display technology. Protein libraries of mutant
neurolysins composed of 120 species were displayed on the
surface and screening was performed using two fluorescencequenching peptides, the matrix metalloproteinase-2/9- (MMPs2/9-) and MMP-3-specific substrates, which consisted of similar
amino acids, except for alanine (for MMPs-2/9) or glutamic acid
(for MMP-3) at the specific amino acid position. As a result,
although wild-type neurolysin effectively cleaved the MMPs2/9-specific substrate, the Y610L (Tyr610Leu) mutant
neurolysin showed a marked change in substrate specificity,
namely that it preferred the MMP-3-specific substrate. Because
peptidases have various important functions in regulating
bioactive peptides, screening of drug candidates, and creating
peptides for oral nutrition supplements, etc., the tailor-made
modification of substrate specificity assisted by molecular
display technology is valuable.

5 Summary
In this review, we described the principle of molecular display
technology of yeast and some of its major achievements. In
addition to the work introduced here, very attractive applications of
molecular display on yeast, including a morphological study,115 the
development of an oral vaccine116 and a study of signal transduction
using receptor-engineered cells,117 have also been reported.
Engineered yeast cells displaying proteins on their surfaces
were named arming yeast when introduced in 1997.118 To
utilize these arming yeast thoroughly, the development of
analytical devices is indispensable. It is also true that molecular
display technology will contribute to the progress of single-cell
analysis. For instance, a microchamber array is a potentially
powerful apparatus to screen positive clones from large-scale
libraries constructed by molecular display technology.119,120
Both cells and devices are in a complementary relationship with
each new development. Although the yeast S. cerevisiae has
usually been used as a eukaryotic model cell for biological
experiments because of the ease of its manipulation and its
genetic background, molecular display technology using the
same yeast is becoming a leading technique in biotechnology,
and arming yeasts will be model cells in various methods of
analytical science in the near future.

48

ANALYTICAL SCIENCES JANUARY 2009, VOL. 25

6 References
33.
1. C. A. Janeway, P. Travers, M. Walport, and M. J. Shlomchik,
Immunobiology, 6th ed., 2005, Garland Science, New York.
2. B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and
P. Walter, Molecular Biology of the Cell, 4th ed., 2002,
Garland Science, New York.
3. G. P. Smith, Science, 1985, 228, 1315.
4. J. K. Scott and G. P. Smith, Science, 1990, 249, 386.
5. H. R. Hoogenboom, A. D. Griffiths, K. S. Johnson, D. J.
Chiswell, P. Hudson, and G. Winter, Nucleic Acids Res.,
1991, 19, 4133.
6. I. Fujii, S. Fukuyama, Y. Iwabuchi, and R. Tanimura, Nat.
Biotechnol., 1998, 16, 463.
7. G. Georgiou, H. L. Poetschke, C. Stathopoulos, and J. A.
Francisco, Trends Biotechnol., 1993, 11, 6.
8. M. Little, P. Fuchs, F. Breitling, and S. Dbel, Trends
Biotechnol., 1993, 11, 3.
9. J. A. Francisco, C. Stathopoulos, R. A. Warren, D. G. Kilburn,
and G. Georgiou, Biotechnology [NY], 1993, 11, 491.
10. J. Maurer, J. Jose, and T. F. Meyer, J. Bacteriol., 1997, 179,
794.
11. A. Wentzel, A. Christmann, T. Adams, and H. Kolmar, J.
Bacteriol., 2001, 183, 7273.
12. L. Hedegaard and P. Klemm, Gene, 1989, 85, 115.
13. B. Westerlund-Wikstrm, Int. J. Med. Microbiol., 2000,
290, 223.
14. S. Sthl, A. Robert, E. Gunneriusson, H. Wernrus, F. Cano,
S. Liljeqvist, M. Hansson, T. N. Nguyen, and P. Samuelson,
Int. J. Med. Microbiol., 2000, 290, 571.
15. P. Samuelson, F. Cano, A. Robert, and S. Sthl, FEMS
Microbiol. Lett., 1999, 179, 131.
16. J. Narita, K. Okano, T. Kitao, S. Ishida, T. Sewaki, M. H.
Sung, H. Fukuda, and A. Kondo, Appl. Environ. Microbiol.,
2006, 72, 269.
17. J. C. Piard, I. Hautefort, V. A. Fischetti, S. D. Ehrlich, M.
Fons, and A. Gruss, J. Bacteriol., 1997, 179, 3068.
18. E. Bernasconi, J. E. Germond, M. Delley, R. Fritsch, and
B. Corthsy, Appl. Environ. Microbiol., 2002, 68, 2917.
19. M. Ueda and A. Tanaka, Biotechnol. Adv., 2000, 18, 121.
20. M. Ueda and A. Tanaka, J. Biosci. Bioeng., 2000, 90,125.
21. A. Kondo and M. Ueda, Appl. Microbiol. Biotechnol., 2004,
64, 28.
22. G. H. Fleet, The Yeasts, ed. A. H. Rose and J. S. Harrison,
2nd ed., 1991, Vol. 4, Academic Press, 199.
23. D. J. Manners, A. J. Masson, and J. C. Patterson, Biochem.
J., 1973, 135, 19.
24. D. J. Manners, A. J. Masson, and J. C. Patterson, Biochem.
J., 1973, 135, 31.
25. M. Horisberger and M. Vonlanthen, Arch. Microbiol., 1977,
15, 62.
26. V. J. Cid, A. Duran, F. D. Rey, M. P. Snyde, C. Nombela,
and M. Sanchez, Microbiol. Rev., 1995, 59, 345.
27. E. Valentin, E. Herrero, F. I. J, Pastor, and R. Sentandreu, J.
Gen. Microbiol., 1984, 130, 1419.
28. G. H. Fleet and D. J. Manners, J. Gen. Microbiol., 1997, 98,
315.
29. K. G. Hardwick, J. C. Boothroyd, A. D. Rudner, and H. R.
B. Pelham, EMBO J., 1992, 11, 4187.
30. P. N. Lipke, D. Wojciechowicz, and J. Kurjan, Mol. Cell.
Biol., 1989, 9, 3155.
31. J. M. Van der Vaart, L. H. P. Caro, J. W. Chapman, F. M.
Klis, and C. T. Verrips, J. Bacteriol., 1995, 177, 3104.
32. J. Watari, Y. Takata, M. Ogawa, H. Sahara, M. Koshino,

34.

35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.

58.
59.

M.-L. Onnela, U. Airaksinen, R. Jaatinen, M. Penttila, and

S. Keranen, Yeast, 1994, 10, 211.
A. Roy, C. F. Lu, D. L. Marykwas, P. N. Lipke, and J.
Kurjan, Mol. Cell. Biol., 1991, 11, 4196.
R. Schekman and P. Novick, The Molecular Biology of the
Yeast Saccharomyces, ed. J. N. Strathern, E. W. Jpnes, and
J. R. Broach, 1982, Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York, 361.
R. Schekman, Curr. Opin. Cell Biol., 1992, 4, 587.
J. C. Kapteyn, R. C. Montijn, E. Vink, J. de la Crua, A.
Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M.
Klis, Glycobiology, 1996, 6, 337.
C. F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol., 1994,
14, 4825.
C. F. Lu, R. C. Montijin, J. L. Brown, F. M. Klis, J, Kurjan,
H. Bussey, and P. N. Lipke, J. Cell. Biol., 1995, 128, 333.
B. L. Miki, N. H. Poon, A. P. James, and V. L. Seligy, J.
Bacteriol., 1982, 150, 878.
A. W. Teunissen, E. Holub, J. van der Hucht, J. A. van den
Berg, and H. Y. Steensman, Yeast, 1993, 9, 423.
N. Sato, T. Matsumoto, M. Ueda, A. Tanaka, H. Fukuda,
and A. Kondo, Appl. Microbiol. Biotechnol., 2002, 60, 469.
T. Matsumoto, H. Fukuda, M. Ueda, A. Tanaka, and A.
Kondo, Appl. Environ. Microbiol., 2002, 68, 4517.
C. Cappellaro, K. Hauser, V. Mrsa, M. Watzele, G. Watzele,
C. Gruber, and W. Tanner, EMBO J., 1991, 10, 4081.
E. T. Boder and K. D. Wittrup, Nat. Biotechnol., 1997, 15,
553.
M. C. Keike, E. V. Shusta, E. T. Boder, D. M. Kranz, and K.
D. Wittrup, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 5651.
S. Ueda and Y. Kobe, J. Ferment. Technol., 1980, 58, 237.
S. Hayashida and P. Q. Flor, Agric. Biol. Chem., 1982, 46,
1639.
T. Ashikari, N. Kiuchi-Goto, Y. Tanaka, Y. Shibano,
T. Amachi, and H. Yoshizumi, Appl. Microbiol. Biotechnol.,
1989, 30, 515.
T. Ashikari, S. Kunisaki, N. Matsumoto, T. Amachi, and
H. Yoshizumi, Appl. Microbiol. Biotechnol., 1989, 32, 129.
T. Ashikari, N. Nakamura, Y. Tanaka, N. Kiuchi, Y. Shibano,
T. Tanaka, T. Amachi, and H. Yoshizumi, Agric. Biol.
Chem., 1986, 50, 957.
T. Murai, M. Ueda, M. Yamamura, H. Atomi, Y. Shibasaki,
N. Kamasawa, M. Osumi, T. Amachi, and A. Tanaka, Appl.
Environ. Microbiol., 1997, 63, 1362.
H. Shigechi, K. Uyama, T. Matsumoto, M. Ueda, A. Tanaka,
H. Fukuda, and A. Kondo, J. Mol. Catal. B: Enzym., 2002,
17, 179.
R. Nakajima, T. Imanaka, and S. Aiba, J. Bacteriol., 1985,
163, 401.
T. Murai, M. Ueda, Y. Shibasaki, N. Kamasawa, M. Osumi,
T. Imanaka, and A. Tanaka, Appl. Microbiol. Biotechnol.,
1999, 51, 65.
T. Murai, M. Ueda, H. Atomi, Y. Shibasaki, N. Kamasawa,
M. Osumi, T. Kawaguchi, M. Arai, and A. Tanaka, Appl.
Microbiol. Biotechnol., 1997, 48, 499.
Y. Fujita, S. Takahashi, M. Ueda, A. Tanaka, H. Okada,
Y. Morikawa, T. Kawaguchi, M. Arai, H. Fukuda, and
A. Kondo, Appl. Environ. Microbiol., 2002, 68, 5136.
M. Kaya, J. Ito, A. Kotaka, K. Matsumura, H. Bando, H.
Sahara, C. Ogino, S. Shibasaki, K. Kuroda, M. Ueda,
A. Kondo, and Y. Hata, Appl. Microbiol. Biotechnol., 2008,
79, 51.
M. P. Schreuder, A. T. A. Mooren, H. Y. Toschka, C. T.
Verrips, and F. M. Klis, Trends Biotechnol., 1996, 14, 115.
M. Washida, S. Takahashi, M. Ueda, and A. Tanaka, Appl.

ANALYTICAL SCIENCES JANUARY 2009, VOL. 25

Microbiol. Biotechnol., 2001, 56, 681.
60. T. Matsumoto, M. Ito, H. Fukuda, and A. Kondo, Appl.
Microbiol. Biotechnol., 2004, 64, 481.
61. M. Kato, J. Fuchimoto, T. Tanino, A. Kondo, H. Fukuda,
and M. Ueda, Appl. Microbiol. Biotechnol., 2007, 75, 549.
62. T. Tanino, T. Ohno, T. Aoki, H. Fukuda, and A. Kondo,
Appl. Microbiol. Biotechnol., 2007, 75, 1319.
63. K. Kuroda, S. Shibasaki, M. Ueda, and A. Tanaka, Appl.
Microbiol. Biotechnol., 2001, 57, 697.
64. K. Kuroda, M. Ueda, S. Shibasaki, and A. Tanaka, Appl.
Microbiol. Biotechnol., 2002, 59, 259.
65. P. Bossier, P. Goethals, and C. Rodrigues-Pousada, Yeast,
1997, 13, 717.
66. S. Yaguchi, K. Mitsui, H. Iha, and K. Tsurugi, FEMS
Microbiol. Lett., 2000, 187, 179.
67. T. R. Butt and D. J. Ecker, Microbiol. Rev., 1987, 51, 351.
68. K. Kuroda and M. Ueda, Appl. Microbiol. Biotechnol.,
2006, 70, 458.
69. E. J. Routledge and J. P. Sumpter, J. Biol. Chem., 1997,
272, 3280.
70. J. Nishikawa, K. Saito, J. Goto, F. Dakeyama, M. Matsuo,
and T. Nishihara, Toxicol. Appl. Pharmacol., 1999, 154, 76.
71. P. Sohoni and J. P. Sumpter, J. Endocrinol., 1998, 158, 327.
72. M. Yasui, S. Shibasaki, K. Kuroda, M. Ueda, N. Kawada, J.
Nishikawa, T. Nishihara, and A. Tanaka, Appl. Microbiol.
Biotechnol., 2002, 59, 329.
73. B. Nilsson, T. Moks, B. Jansson, L. Abrahmsn, A.
Elmblad, E. Holmgren, C. Henrichson, T. A. Jones, and M.
Uhln, Protein Eng., 1987, 1, 107.
74. Y. Nakamura, S. Shibasaki, M. Ueda, A. Tanaka, H.
Fukuda, and A. Kondo, Appl. Microbiol. Biotechnol., 2001,
57, 500.
75. S. Shibasaki, A. Kawabata, J. Ishii, S. Yagi, T. Kadonosono,
M. Kato, N. Fukuda, A. Kondo, and M. Ueda, Appl.
Microbiol. Biotechnol., 2007, 75, 821.
76. N. Fukuda, J. Ishii, S. Shibasaki, M. Ueda, H. Fukuda, and
A. Kondo, Appl. Microbiol. Biotechnol., 2007, 76, 151.
77. H. Miyashita, Y. Karaki, M. Kikuchi, and I. Fujii, Proc.
Natl. Acad. Sci. U. S. A., 1993, 90, 5337.
78. Y. Lin, S. Shiraga, T. Tsumuraya, T. Matsumoto, A. Kondo,
I. Fujii, and M. Ueda, J. Mol. Catal. B: Enzym., 2004, 28, 241.
79. Y. Lin, S. Shiraga, T. Tsumuraya, I. Fujii, T. Matsumoto, A.
Kondo, and M. Ueda, J. Mol. Catal. B: Enzym., 2004, 28, 247.
80. C. Y. Huang, A. L. Bredemeyer, L. M. Walker, C. H.
Bassing, and B. P. Sleckman, Eur. J. Immunol., 2008, 38, 342.
81. B. Westermann and W. Neupert, Yeast, 2000, 16, 1421.
82. M. Dehio, A. Knorre, C. Lanz, and C. Dehio, Gene, 1998,
215, 223.
83. K. Ye, S. Shibasaki, M. Ueda, T. Murai, N. Kamasawa, M.
Osumi, K. Shimizu, and A. Tanaka, Appl. Microbiol.
Biotechnol., 2000, 54, 90.
84. T. T. Yang, L. Cheng, and S. R. Kain, Nucleic Acids Res.,
1996, 24, 4592.
85. S. Shibasaki, M. Ueda, T. Iizuka, M. Hirayama, Y. Ikeda, N.
Kamasawa, M. Osumi, and A. Tanaka, Appl. Microbiol.
Biotechnol., 2001, 55, 471.
86. K. Umemura, H. Atomi, T. Kanai, Y. Teranishi, M. Ueda,
and A. Tanaka, Appl. Microbiol. Biotechnol., 1995, 43, 489.
87. T. Kanai, H. Atomi, K. Umemura, H. Ueno, Y. Teranishi,
M. Ueda, and A. Tanaka, Appl. Microbiol. Biotechnol.,
1996, 44, 759.
88. S. Shibasaki, M. Ueda, K. Ye, K. Shimizu, N. Kamasawa,
M. Osumi, and A. Tanaka, Appl. Microbiol. Biotechnol.,
2001, 57, 528.

49
89. R. D. Mitra, C. M, Silva, and D. C. Youvan, Gene, 1996,
173, 13.
90. H. Rudolph and A. Hinnen, Proc. Natl. Acad. Sci. U. S. A.,
1987, 84, 1340.
91. S. Harashima, T. Mizuno, H. Mabuchi, S. Yoshimitsu, R.
Ramesh, M. Hasebe, A. Tanaka, and Y. Oshima, Mol. Gen.
Genet., 1995, 247, 716.
92. M. C. Lorenz and J. Heitman, EMBO J., 1998, 17, 1236.
93. A. M. Marini, S. Soussi-Boudekou, S. Vissers, and B.
Andre, Mol. Cell. Biol., 1997, 17, 4282.
94. S. Shibasaki, Y. Ninomiya, M. Ueda, M. Iwahashi, T.
Katsuragi, Y. Tani, S. Harashima, and A. Tanaka, Appl.
Microbiol. Biotechnol., 2001, 57, 702.
95. L. Poppenborg, K. Friehs, and E. Flaschel, J. Biotechnol.,
1997, 58, 79.
96. J. H. Cha, C. F. Wu, J. J. Valdes, G. Rao, and W. E. Bentley,
Biotechnol. Bioeng., 2000, 67, 565.
97. T. P. St. John and R. W. Davis, J. Mol. Biol., 1981, 152, 285.
98. C. C. Contaxis and F. J. Reithel, Biochem. J., 1971, 124, 623.
99. G. R. Adolf, B. Fruhbaeis, R. Hauptmann, I. Kalsner, I. M.
Fogy, E. Ostermann, E. Patzelt, R. Schwendenwein, W.
Sommergruber, and A. Zophel, Biochim. Biophys. Acta,
1991, 1089, 167.
100. S. Shibasaki, A. Tanaka, and M. Ueda, Biosens.
Bioelectron., 2003, 19, 123.
101. S. Shibasaki, K. Kuroda, H. Duc Nguyen, T. Mori, W. Zou,
and M. Ueda, Appl. Microbiol. Biotechnol., 2006, 70, 451.
102. S. Bhattacharya, L. Chen, J. R. Broach, and S. Powers,
Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 2984.
103. S. P. Srinivasa, L. S. Bernstein, K. J. Blumer, and M. E.
Linder, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 5584.
104. P. M. Pryciak and F. A. Huntress, Genes Dev., 1998, 12, 2684.
105. S. Gurunathan, M. Marash, A. Weinberger, and J. E. Gerst,
Mol. Biol. Cell, 2002, 13, 1594.
106. S. Fields and O. Song, Nature, 1989, 340, 245.
107. B. Nyfeler, S. W. Michnick, and H. P. Hauri, Proc. Natl.
Acad. Sci. U. S. A., 2005, 102, 350.
108. S. Shibasaki, K. Sakata, J. Ishii, A. Kondo, and M. Ueda,
Appl. Microbiol. Biotechnol., 2008, 80, 735.
109. W. Zou, M. Ueda, and A. Tanaka, Appl. Microbiol.
Biotechnol., 2002, 58, 806.
110. S. Shiraga, M. Ishiguro, H. Fukami, M. Nakao, and M.
Ueda, Appl. Microbiol. Biotechnol., 2005, 68, 779.
111. N. Okochi, M. Kato, T. Kadonosono, and M. Ueda, Appl.
Microbiol. Biotechnol., 2007, 77, 597.
112. S. Paul, D. J. Volle, C. M. Beach, and R. J. Massey, Science,
1989, 244, 1158.
113. T. Kadonosono, M. Kato, and M. Ueda, Protein Eng., Des.
Sel., 2008, 21, 507.
114. F. Checler, J. Vincent, and P. Kitabgi, J. Biol. Chem., 1986,
261, 11274.
115. Y. Shibasaki, N. Kamasawa, S. Shibasaki, W. Zou, T.
Murai, M. Ueda, A. Tanaka, and M. Osumi, FEMS
Microbiol. Lett., 2000, 192, 243.
116. Y. Tamaru, M. Ohtsuka, K. Kato, S. Manabe, K. Kuroda,
M. Sanada, and M. Ueda, Biotechnol. Prog., 2006, 22, 949.
117. J. Ishii, S. Matsumura, S. Kimura, K. Tatematsu, S. Kuroda,
H. Fukuda, and A. Kondo, Biotechnol. Prog., 2006, 22, 954.
118. Anonymous, Chem. Eng. News, 1997, 75, 32.
119. T. Fukuda, S. Shiraga, M. Kato, S. Suye, and M. Ueda,
Biotechnol. Prog., 2006, 22, 944.
120. T. Fukuda, M. Kato, S. Suye, and M. Ueda, J. Biosci.
Bioeng., 2007, 104, 241.