Molecular Display Technology Using Yeast—Arming Technology

41ANALYTICAL SCIENCES JANUARY 2009, VOL. 25 41

2009 The Japan Society for Analytical Chemistry

1 Introduction

11 Overview of molecular displayCells 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

other compounds as signal molecules, and transduction of information into the cytosol or nucleus occurs after ligand-binding.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

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)

To whom correspondence should be addressed.E-mail: [email protected]

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 yeast2 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

23 Display of antibodies and related molecules for diagnostics and therapeutics

3 Sensing and Monitoring 45 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 interactions4 Combinatorial Libraries 465 Summary 476 References 48

42 ANALYTICAL SCIENCES JANUARY 2009, VOL. 25

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

wall with b-1,3- or b-1,6-glucanase.28Many 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 cell-surface 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 Golgi-derived 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 yeastFlo1, Cwps, and a- and a-agglutinins have been utilized for

the display of proteins on a yeast cell surface. Flo1 is a lectin-like cell-wall protein and plays a major role in flocculation.39,40

Fig. 1 Architecture of the yeast cell wall. SMP, glucanase-extractable surface-layer mannoprotein; PP, SDS-extractable periplasmic protein.

Fig. 2 Schematic illustration of GPI anchor. An anchored protein is released with cleaving by phosphatidyl inositol-specific phospholipase C (PI-PLC).

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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 anchor-attachment signal. To display foreign proteins on the cell wall, genetic information of each agglutinin is utilized. The cell wall-anchoring 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 biocatalystsThe 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

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

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.


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

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 component-displaying 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 ion-inducible genetic element, the CUP1 promoter.67 The copper ion-induced aggregation did not interfere with the copper ion-adsorbing 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-


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

sensing environmental changes inside or outside of living cells.

31 Exploration of the cell wall by a visible reporterThe 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-a-agglutinin 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 changesAs 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

Fig. 4 Fab-display on the yeast cell surface.


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 high-phosphate 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 non-invasive manner.

33 Monitoring of the production of bioactive compoundsIn the first application of GFP in bioprocess monitoring,

Poppenborg et al. reported the monitoring of a hisactophilin-GFP 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

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 intra- and 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 membrane-targeting 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

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.


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 fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) 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 MMPs-2/9-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.

Fig. 6 Anchoring of proteins in the cytoplasmic face to observe protein-protein interactions.


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Molecular Display Technology Using Yeast—Arming Technology