Genetic immobilization of proteins on the yeast cell surface

Biotechnology Advances 18 (2000) 121–140

Research review paper

Genetic immobilization of proteins on the yeast cell surface Mitsuyoshi Ueda, Atsuo Tanaka* Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

Abstract A genetic system has been exploited to immobilize proteins in their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae. DNAs encoding proteins with a secretion signal peptide were fused with the genes encoding yeast agglutinins, a- and ␣-type proteins involved in mating. The fusion gene was introduced into S. cerevisiae and expressed under the control of several promoters. Appearance of the fused proteins expressed on the cell surface was demonstrated biochemically and by immunofluorescence and immunoelectron microscopy techniques. ␣-Galactosidase from Cyamopsis tetragonoloba seeds, peptide libraries including scFv and variable regions of the T cell receptor from mammalian cells have been successfully immobilized on the yeast cell wall in the active form. Recently, surface-engineered yeasts have been constructed by immobilizing the enzymes and a functional protein, for example, green fluorescent protein (GFP) from Aequorea victoria. The yeasts were termed ‘arming yeasts’ with biocatalysts or functional proteins. Such arming cells displaying glucoamylase from Rhizopus oryzae and ␣-amylase from Bacillus stearothermophilus, or carboxymethylcellulase and ␤-glucosidase from Aspergillus acleatus, could assimilate starch or cellooligosaccharides as the sole carbon source, although S. cerevisiae cannot intrinsically assimilate these substrates. GFP-arming cells can emit green fluorescence from the cell surface in response to the environmental conditions. The approach described in this review will enable us to endow living cells, including yeast cells, with novel additional abilities and to open new dimensions in the field of biotechnology. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Cell surface engineering; Surface display of proteins; Agglutinin; GPI-anchor; Anchoring

1. Introduction Conventional methods for immobilization of proteins through covalent bonds have some merits. For example, the proteins are immobilized through strong bonds and the levels of dissociation are low in substrate or salt solutions even at high concentrations. However, changes in structure of the immobilized proteins or in their characteristics often occur, due to severe * Corresponding author. Tel.: ⫹81-75-753-5524; fax: ⫹81-75-753-5534. E-mail address: [email protected] (A. Tanaka) 0734-9750/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0 7 3 4 - 9 7 5 0 ( 0 0 ) 0 0 0 3 1 -8

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treatment and there are many difficulties in the determination of the immobilization reaction conditions. Alternative immobilization methods (e.g. through ionic interactions) can be less damaging to protein structure and function, but the resultant immobilized proteins are easily dissociated. The application of genetic engineering, using the cell surface as a carrier for protein immobilization, can overcome the limitations of the above-described covalent coupling approach. The proteins can be regenerated according to the activation of the promoter, and they are ‘naturally’ immobilized on the cell surface. Surface proteins are responsible for most cell surface functions, serving as cell–cell adhesion molecules, specific receptors, enzymes, transport proteins, and so on. Some surface proteins extend across the plasma membrane, and other surface proteins are bound by noncovalent or covalent interaction to cell surface components. Cells have intrinsic systems for anchoring surface-specific proteins and for confining surface proteins to particular domains on the cell surface. In terms of application, the function of the cell surface, which can still be considered an unexploited area, should be reassessed, since many of the mechanisms for protein transport to the cell surface are now known. Establishment of these systems to immobilize heterologous proteins on the cell surface of microorganisms is expected to be useful for the segregation of produced polypeptides, construction of microbial biocatalysts, whole-cell adsorbents, and live vaccines, etc. Immobilization of enzymes on the cell surface saves tedious purification process of enzymes to be used in conventional immobilization. Expression of proteins on the cell surface of Saccharomyces cerevisiae would offer more advantages than other microbial cells. First, as S. cerevisiae is widely used in industrial production of proteins and chemicals, enzyme-coated yeast cells could be used as novel whole-cell biocatalysts, because surface-immobilized proteins are covalently linked to glucan in the cell wall, rendering them resistant to extraction. Second, as S. cerevisiae is safe (GRAS, generally regarded as safe) for oral use, it can be used in food and pharmaceutical products. Utilization of the cell surface of living cells is also attractive for many applications in microbiology and molecular biology.

2. Fundamental studies on the cell surface of yeast The yeast S. cerevisiae has a rigid thick cell wall of about 200 nm in thickness outside of the plasma membrane. The cell wall of S. cerevisiae is mainly composed of mannoproteins and ␤-linked glucans (Fleet, 1991), and has a bilayered structure consisting of an internal skeletal layer of glucan, composed of ␤-1, 3- and ␤-1, 6-linked glucose (Manners et al., 1973a, b), and a fibrillar or brush-like outer layer composed predominantly of mannoproteins (Horisberger and Vonlanthen, 1977). These proteins are linked to glucan through covalent bonds (Fig. 1). Two types of mannoproteins are present in the cell wall of S. cerevisiae (Cid et al., 1995; Klis, 1994). Mannoproteins loosely associated with the cell wall through non-covalent bonds are extractable with sodium dodecylsulfate (SDS). When the isolated yeast cell wall is solubilized with hot SDS, about 60 proteins are released (Valentin et al., 1984). The other types of mannoproteins are extractable by glucanase, which are released only by ␤-1, 3- or ␤-1, 6-glucanase digestion of the glucan layer of the cell wall (Fleet and Manners, 1977). Among these glucanase-extractable mannoproteins on the cell surface of S. cerevisiae, the

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Fig. 1. Structure of the cell surface of S. cerevisiae. CW, cell wall; PM or CM, plasma membrane or cell membrane; PMP, plasma membrane protein; PP, SDS-extractable periplasmic protein; SMP, glucanase-extractable surface-layer mannoprotein.

mating-type specific agglutinins (Lipke and Kurjan, 1992) mediate the direct cell–cell adhesion between cells of the opposite type during mating. These agglutinins represent minor cell wall components which are presumably located on the outermost surface. Mating type a and ␣ cells express a-agglutinin and ␣-agglutinin, respectively (Terrance et al., 1987; Watzele et al., 1988). ␣-Agglutinin is encoded by the AG␣1 gene (Lipke et al., 1989) and interacts with the binding subunit of the agglutinin complex of a-type cells (Cappellaro et al., 1991). a-Agglutinin consists of a core subunit (Aga1p) encoded by the AGA1 gene (Roy et al., 1991) that is linked through disulfide bridges to a small binding subunit (Aga2p) encoded by the gene AGA2 (Cappellaro et al., 1991). The structures of both ␣-agglutinin and the core subunit of a-agglutinin are composed of a secretion signal region, an active region, a support region rich in serine and threonine, and a putative glycosylphosphatidylinositol (GPI) anchor-attachment signal (Fig. 2). The proteins presumably occur in a heavily O-glycosylated form (Lipke et al., 1989; Roy et al., 1991; Wojciechowicz et al., 1993). GPI anchors were found on many eukaryotic plasma membrane proteins, ranging from coat proteins of protozoa to mammalian cell-adhesion molecules (Cross, 1990; Fredette et al., 1993; Dustin et al., 1987; Homans et al., 1988). The structure of the GPI anchor is highly conserved among molecules from various eukaryotic organisms (Ferguson and Williams, 1988). The core structure of the yeast GPI anchor is similar to that found in other eukaryotes (Conzelmann et al., 1988; Lipke et al., 1989; Leidich et al., 1994), and is composed of ethanolamine phosphate (6)mannose(␣1,2)mannose(␣1,6)mannose(␣1,4)glucosamine(␣1,6)inositol phospholipid (Fig. 3). The lipid composition varies among yeast GPI anchors. Many cell surface proteins in yeast, such as Ag␣1 (Lipke et al., 1989), Aga1 (Roy et al., 1991), Flo1 (Watari et al., 1994), Sed1 (Hardwick et al., 1992), and Cwp1, Cwp2, Tip1, Tir1/

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Fig. 2. Structure of ␣-agglutinin. The predicted cleavage site, residing in the C-terminal region containing a GPI anchor-attachment signal sequence, is indicated by arrows. The predicted ␻ site in the text is glycine (G).

Srp1 (Van der Vaart et al., 1995), have GPI anchors, which play important roles for surface expression of cell-surface proteins and are essential for the viability of yeasts. These glycophospholipid moieties are covalently attached to the C-terminal region of proteins and their primary function is to afford the stable association of proteins with the membrane. GPIanchored proteins contain hydrophobic peptides at their C-terminus. After the completion of protein synthesis, the precursor protein remains anchored in the endoplasmic reticulum (ER) membrane by the hydrophobic carboxyl-terminal sequence, with the rest of the protein in the ER lumen. Within less than a minute, the hydrophobic carboxyl-terminal sequence is cleaved

Fig. 3. General structure of a GPI anchor. Man, Mannose; GlcNH2, glucosamine.

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at the ␻ site and concomitantly replaced with the GPI anchor presumably by a transamidase. Because of the covalently linked lipid anchor, the protein remains membrane-bound, exposed initially on the luminal side of the ER and eventually on the cell exterior. The localization of both a-agglutinin and ␣-agglutinin at the cell surface occurs through the secretory pathway (Tohoyama and Yanagishima, 1985, 1987). Protein secretion in S. cerevisiae comprises transfer through various membrane-enclosed compartments constituting the secretory pathway (Fig. 4). Secreted proteins are first translocated into the lumen of the ER, and then transported from the ER to the Golgi apparatus and from there to the plasma membrane in membrane-enclosed vesicles (Schekman and Novick, 1982; Schekman, 1992). Fusion of the Golgi-derived secretory vesicles with the plasma membrane releases the secreted proteins to the cell exterior. Post-translational proteolytic modification of precursors of secretory peptides occurs in the late compartments of the secretory pathway (trans cisternae of the Golgi apparatus and secretory vesicles). The Kex2 endopeptidase is located in the trans cisternae of the Golgi apparatus in S. cerevisiae to remove the proregion of the precursors (e.g. ␣-factor pheromone, etc.) (Wagner et al., 1987). ␣-Agglutinin was proposed to be further transported to the outside of the plasma membrane through the general secretory pathway in a GPI-anchored form and then released from the plasma membrane by phosphatidylinositol-specific phospholipase C (PI-PLC) and transferred to the outermost surface of the cell wall (Lu et al., 1994). Cell wall anchorage of ␣-agglutinin is accomplished by addition of ␤1,6-glucan to the GPI anchor remnant of ␣-agglutinin, in a manner dependent on prior addition of a GPI anchor to ␣-agglutinin (Lu et al., 1995; Kapteyn et al., 1996).

Fig. 4. Illustration of transport of ␣-agglutinin to the yeast cell surface. ER, endoplasmic reticulum; PI-PLC, phosphatidylinositol-specific phospholipase C.

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3. General principle of immobilization of proteins on the cell surface of yeast Flocculins, encoded by FLO1, Cwps and a- and ␣-agglutinins, have been utilized for immobilization of proteins on the cell surface of yeast. In this context, molecular information of ␣-agglutinin and Aga2p of a-agglutinin has been available for target heterologous proteins to the outmost surface of the glycoprotein layer of the cell wall. In the latter case, the C-terminal fusion to the Aga2p has been accomplished, whereby the signal sequence of Aga2p was used as seen in Fig. 5. Binding to Aga1p was carried out with disulfide bonds between Aga1p and Aga2p. On the other hand, the anchoring signal of ␣-agglutinin has been combined with the signal of the secreted enzymes, using genetic engineering techniques. The scheme in Fig. 6 shows the general structure of the gene for cell surface immobilization of an enzyme in such an ␣-agglutinin construct. The C-terminal half of ␣-agglutinin (320 amino acid residues, CH-Ag␣p) contains a glycosylphosphatidylinositol (GPI) anchor attachment signal at the C-terminal end. Like other cell surface proteins, this signal is utilized as an anchoring domain for heterologous proteins, since these proteins are covalently linked with glucan.

Fig. 5. Systems for display of heterologous proteins on the cell surface of yeast using ␣-agglutinin (A) and a-agglutinin (B) structures. CH-Ag␣p, C-terminal half of ␣-agglutinin; Aga1p, core subunit encoded by the AGA1 gene; Aga2p, small subunit encoded by the AGA2 gene.

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Fig. 6. Molecular design for display of proteins on the cell surface of yeast using ␣-agglutinin structure. (A) Design of gene; (B) model of translated protein. C-Terminal half of ␣-agglutinin is a domain rich in serine and threonine as illustrated in Fig. 2.

4. Genetic immobilization of enzymes and functional proteins on the cell surface of yeast 4.1. Genetic immobilization of ␣-galactosidase ␣-Galactosidase from Cyamopsis tetragonoloba seeds, as a reporter enzyme, was the first heterologous enzyme targeted to the cell wall of S. cerevisiae in the active form (Schreuder et al., 1993, 1996). The invertase signal sequence was used to express the ␣-galactosidaseencoding gene fused to the gene encoding the C-terminal half of ␣-agglutinin. The resultant constructed gene on a multicopy plasmid was expressed under the control of the constitutive phosphoglycerokinase (PGK) promoter. Subcellular fractionation of exponential-phase cells indicated that 70% of the fusion protein incorporated into the cell wall was tightly linked to glucan. Immunofluorescent labeling using polyclonal antiserum demonstrated that the fusion protein was present on the surface-engineered yeast cells. However, the amount of detectable fusion protein varied extensively in each cell. Such a phenomenon was shown to depend on the copy number of the plasmid. The property of the immobilized ␣-galactosidase was evaluated by inhibition with Hg2⫹ and the regeneration of enzymatic activity. 4.2. Genetic immobilization of glucoamylase In the conventional procedure to convert starchy materials to ethanol in high yield, the crude material is cooked at 140–180⬚C prior to amylolysis, and the energy consumed in this process results in high production costs (Maiorella, 1985). To save the energy in the cooking process, pioneering studies were conducted using enzymes which degrade raw starchy materials (Ueda and Koba, 1980; Hayashida and Flor, 1982). Further work has led to the development of a fermentation system using the enzymes prepared from Rhizopus oryzae, which efficiently digests raw starch in cereal grits (Matsumoto et al., 1982). Thereafter, intensive research has been conducted to construct further improved starch-utilizing systems, employ-

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ing yeast strains prepared by genetic engineering to produce secretory amylolytic enzymes (Tubb, 1986). Construction of yeast strains to secrete glucoamylase from R. oryzae has been attempted for application to fermentation of raw starch without cooking (Ashikari et al., 1986, 1989a, b). Glucoamylase from R. oryzae is an exo-type amylolytic enzyme that cleaves effectively ␣-1,4-linked and ␣-1,6-linked glucose from starch. The engineered yeast cells, containing immobilized glucoamylase on the cell surface, may be able to saccharify starch by glucoamylase on its cell wall and assimilate the released glucose to proliferate and ferment (Fig. 7). Novel yeast cells were prepared that harbored a multicopy plasmid to express the glucoamylase/␣-agglutinin fusion gene containing the secretion signal sequence of the glucoamylase under the control of the GAPDH promoter. The resulting yeasts were demonstrated to exhibit the cell-associated glucoamylase activity without secretion of the active enzyme when cultivated in medium containing 2% glucose. The amount of displayed glucoamylase induced by glucose was much higher than that induced by pheromone (Table 1). Even after extraction from cell wall (with hot SDS) of non-covalently bound glucoamylase or the protein bound through disulfide bridges, 93% of the total extractable glucoamylase protein could still be removed by glucanase, indicating that the glucoamylase was covalently attached to the cell wall. Cultivation on an agar plate demonstrated that the engineered cells hydrolyzed starch and produced a halo strictly around the colony, while no halo formation was observed around the parent cells (Murai et al., 1997a). Immunofluorescent labeling of cells with anti-glucoamylase IgG showed that the cells expressing the glucoamylase/␣-agglutinin fusion gene were uniformly labeled. The intensity, however, varied from cell to cell, probably due to differences in expression levels among the individual cells. By immunoelectron microscopy, gold particles were detected only on the surface of the engineered cells, while few gold particles were detected in the case of the parent cells (Fig. 8).

Fig. 7. A model of cell surface-engineered S. cerevisiae (arming yeast), displaying hydrolytic enzymes. E, Hydrolytic enzymes.

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Table 1 Comparison of the approximate amount of the displayed proteins on the cell surface of yeast Cell surface protein

Amount expressed (molecules/cell)

Ratio (-fold)

␣-Agglutinin Pheromone-induced ␣-Agglutinin Displayed Glucoamylase

ca. 1 ⫻ 103

1

ca. 2 ⫻ 104

20

ca. 6 ⫻ 105

600

The thermal stability characteristics, optimal temperature, and optimal pH of glucoamylase immobilized on the cell surface were evaluated by comparing with those of the secreted free enzyme. The activity of the anchored glucoamylase was stable in the temperature range of 0–45⬚C. The optimal temperature and the optimal pH of the anchored glucoamylase were 50⬚C and 4.5, respectively. While no differences in thermal stability and pH optima were observed between anchored and free glucoamylases, the optimal temperature of anchored glucoamylase was somewhat lower than that of the free enzyme (Ueda et al., 1998). When cells were cultivated aerobically with 1% soluble starch as the sole carbon source, the engineered yeasts proliferated to essentially the same level as that of yeasts cultured on 1% glucose. In contrast, no growth was observed with the control yeasts. These data showed that the cell surface-immobilized glucoamylase catalyzed sufficiently the hydrolysis of starch. The glucoamylase/␣-agglutinin fusion gene-integrated cells exhibited glucoamylase activity on the cell surface as well as the cells harboring multicopy plasmid. The cells were capable of utilizing starch as the sole source of carbon and energy. Following repeated (three times) cultivation for 120 h, the mitotic stability of the cells was much higher than that of the cells harboring multicopy plasmid (Ueda et al., 1998). 4.3. Genetic co-immobilization of glucoamylase and ␣-amylase For cell-surface display of ␣-amylase (EC 3.2.1.1), an endo-type amylolytic enzyme from Bacillus stearothermophilus CU21 (BSTA) (Nakajima et al., 1985) that cleaves ␣-1,4-glycosidic linkages, the BSTA/C-terminal half of ␣-agglutinin fusion gene was integrated into the chromosome of S. cerevisiae. In this case, about 52% of the total ␣-amylase activity was detected in the culture supernatant, due to proteolytic processing of the fusion protein. The deduced amino acid sequence of BSTA indicated one possible processing site for the Kex2 endopeptidase, Val-Pro-Arg, at amino acid residues 481–483. Kex2, one of the endopeptidases in the secretory process of ␣-mating factor and killer toxin precursors, exhibits substrate specificity toward carboxyl sites of Lys-Arg, Arg-Arg and Pro-Arg sequences (Mizuno et al., 1989). A Kex2-resistant BSTA/␣-agglutinin fusion protein was designed by eliminating 33 residues from the C-terminus of BSTA. Cells harboring the C-terminal truncated BSTA/␣-agglutinin fusion gene exhibited ␣-amylase activity only in the cell pellet fraction. On plate assays, a large halo was detected around the colony of cells which secreted ␣-amylase, while small halo formation was observed strictly around cells in which the C-terminal truncated BSTA was immobilized, as in the case of glucoamylase-displaying cells (Murai et al., 1999).

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Fig. 8. Immunofluorescent labeling and immunoelectron micrograph of surface-engineered cells (arming yeast) displaying glucoamylase (A) and CM-cellulase (B) on the cell surface. Nomarsky differential interference micrographs (A,D); immunofluorescence micrographs (b,e). Immunoelectron micrograph using gold-labeled antibody against glucoamylase (c) and CM-cellulase (f). CW, cell wall; M, mitochondrion. Arrowheads: gold particles (diameter, 5 nm).

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For co-immobilization of glucoamylase and ␣-amylase on cell surfaces, both the glucoamylase/␣-agglutinin gene and the C-terminal truncated ␣-amylase/␣-agglutinin gene were integrated into the yeast chromosomes. Such cells exhibited both glucoamylase and ␣amylase activities only in the cell pellet fraction. Immunofluorescent labeling of cells with anti-glucoamylase IgG and FITC-goat IgG to rabbit IgG as the second antibody showed the expression of both glucoamylase and ␣-amylase on the cell surface of the same cell (Ueda et al., 1998). This was confirmed by the observation of halo formation of the cells grown on a starch-containing plate. When the surface-engineered yeast cells were cultivated aerobically in medium containing 1% soluble starch as the sole carbon source, the growth of the cells reached the same level as cells cultured on 1% glucose. No growth on starch was observed with the parent cells. The engineered yeast cells that harbored co-immobilized glucoamylase and ␣-amylase could grow faster than the glucoamylase-immobilized cells. ␣-Amylase is an endoglucanase that hydrolyzes starch in a random fashion, producing oligosaccharides. Therefore, the cooperative and sequential reaction probably resulted in the increased rate of glucose formation through the increased production of molecules with nonreducing ends by ␣-amylase, which in turn could serve as substrate molecules for glucoamylase (Murai et al., 1998a, 1999). 4.4. Genetic immobilization of CM-cellulase Cellulose is a linear polymer, consisting of glucose units linked together by ␤-1,4-glycosidic bonds. It is the most abundant carbohydrate in the biosphere. The estimated worldwide rate of cellulose synthesis is approximately 4 ⫻ 107 tons per year. For a long-range solution to resources problems of energy, chemicals, and food, cellulose is the most promising renewable carbon source that is available in large quantities. However, the yeast S. cerevisiae is unable to utilize cellulosic materials in spite of its versatility in industrial fermentation. Because enzymatic hydrolysis of cellulose has the potential to surmount many of the drawbacks of acid hydrolysis, attempts to genetically display cellulolytic enzymes in their active form on the cell surface has been done by constructing a novel cellulose-utilizing yeast, S. cerevisiae. As one of the target enzymes, carboxymethylcellulase (CM-cellulase) from Aspergillus aculeatus, classified as an endo-1,4-␤-D-glucan glucohydrolase (endoglucanase) that cleaves the ␤-1,4-glycosidic linkage of cellulose, was selected. For displaying CM-cellulase on the yeast cell surface, a multicopy plasmid was constructed for expression of the CM-cellulase/␣-agglutinin fusion gene. The construct contained the secretion signal sequence of the CM-cellulase under control of the GAPDH promoter. A plate assay was employed, using Congo red staining on plates containing carboxymethylcellulose, which showed that the engineered cells exhibited cell-associated CM-cellulase activity without secretion of the active enzyme. Upon treatment with SDS, the CM-cellulase was proved to be covalently attached to the cell wall. From the results of immunofluorescent labeling of cells, using anti-CM-cellulase IgG and FITC-goat IgG to rabbit IgG as the second antibody, combined with immunoelectron microscopic observation, the expressed CM-cellulase/␣-agglutinin fusion protein was shown to be anchored on the cell wall of the engineered cells (Fig. 8) (Murai et al., 1997b).

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4.5. Co-immobilization of CM-cellulase and ␤-glucosidase Because CM-cellulase is an endo-type cellulase, the enzymatic degradation of cellulose to glucose requires synergistic hydrolysis by different types of cellulolytic enzymes. It is predicted that short-chain cellooligosaccharides formed by the endo-action of CM-cellulase are converted quickly to glucose by ␤-glucosidase (1,4-␤-D-glucoside glucohydrolase). However, S. cerevisiae lacks a ␤-glucosidase and consequently is unable to utilize cellobiose as the carbon source. Thus, to construct yeast cells, which are able to utilize cellulosic degradation products (i.e. cellooligosaccharides), the display of ␤-glucosidase, in addition to CMcellulase, on cell surface of S. cerevisiae is also necessary. As a vector for coimmobilization of CM-cellulase and ␤-glucosidase from A. aculeatus on the cell surface, a multicopy plasmid for expression of the ␤-glucosidase/␣-agglutinin fusion gene was constructed, that contains the secretion signal sequence of the glucoamylase gene under the control of the GAPDH promoter. The strain harboring this vector showed both CM-cellulase and ␤-glucosidase activities. The parent cells exhibited neither of the activities. Immunofluorescence microscopy, using FITC-goat IgG to rabbit IgG as the second antibody, demonstrated the presence of both CM-cellulase and ␤-glucosidase proteins on the cell surface, when anti-CM-cellulase IgG and anti-␤-glucosidase IgG were used as the primary antibodies. The engineered cells were labeled by fluorescence, using both anti-CM-cellulase IgG and anti-␤-glucosidase IgG, indicating co-display of the CM-cellulase and ␤-glucosidase proteins on the cell surface (Murai et al., 1998b). The engineered yeast cells were cultivated aerobically in medium containing cellobiose as the sole carbon source. The yeast strains harboring both enzyme-coding genes as the multicopy plasmid could grow on cellobiose, while no growth on cellobiose was observed with the parent cells. The surface-engineered yeast cells were cultivated aerobically in the medium supplemented with cellooligosaccharides (approximately 11% (w/w) cellohexaose, 29% (w/w) cellopentaose, 33% (w/w) cellotetraose, 17% (w/w) cellotriose, 4% (w/w) cellobiose, and less than 1% (w/w) glucose) as the sole carbon source. Cell growth was monitored by counting colonies that appeared on YPD plates. The results demonstrated that engineered cells that displayed both enzymes could grow on cellooligosaccharides, while again no growth was observed with the control cells or the cells displaying only the CM-cellulase (Murai et al., 1998b). Much effort has been devoted to utilizing cellulosic materials by employing S. cerevisiae and cellulase complexes from cellulolytic bacteria (Van Rensburg et al., 1996; Wong et al., 1988). However, these attempts have failed because ␤-glucosidase was not expressed. This is the first step for the assimilation of cellulosic materials by a S. cerevisiae strain that expresses heterologous cellulase genes and is also the first report for assimilation of cellooligosaccharides by yeast. The cellulase system of the anaerobic cellulolytic bacterium, Clostridium thermocellum, was shown to naturally produce a discrete multienzyme complex located on the cell surface (Tokatlidis et al., 1991), called cellulosomes (Lamed et al., 1983). The cellulosome is considered to help C. thermocellum to obtain the source of carbon and energy efficiently by enzymatic degradation of cellulose on its cell surface. Considering the advantage of cellulosomes, the displayed enzymes on the cell surface of S. cerevisiae may also facilitate the uptake of glucose liberated on the cell surface. The display of cellulases on

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the cell surface may make it possible to utilize cellulose by yeast. The novel cell surface-engineered cells endowed with a rapid cellooligosaccharide-utilizing ability by displaying two types of cellulolytic enzymes will open a door to develop yeast cells which can perform efficacious fermentation of cellulosic materials. 4.6. Genetic immobilization of peptide libraries Peptide and protein libraries were successfully displayed on the cell surface of yeast using the system of the C-terminal fusion to Aga2p (Boder and Wittrup, 1997; Kieke et al., 1999). Libraries of single-chain Fv (scFv) fragments, displayed on the cell surface of yeast, were screened. The displayed scFv specifically-bound antigen (fluorescein-binding scFv mutant) was selected from the peptide libraries. This result was the first demonstration of a functional antibody fragment displayed on the cell surface of yeast. In addition, the surface display of another molecule similar to antibodies (i.e. the scT cell receptor) was carried out after mutation of the specific variable region residues. Yeast cells, displaying the mutant scTCRs which bound specifically to the peptide/MHC antigen, were selected from the libraries and their properties were evaluated. These results indicated that the display system using the cell surface of yeast could be significant for genetic engineering of T cell receptors by screening of combinatorial peptide libraries. 4.7. Genetic immobilization of green fluorescent proteins (GFP) A visible reporter on the cell surface is an extremely important interface between a cell and its environment. Compared with enzyme-type reporters expressed in cells (Walmsley et al., 1997), visible reporters are powerful and vital markers for gene transcription, membrane protein localization, and ligand binding in molecular and biological analyses. Visible reporters are especially useful, because their monitoring can be carried out at the level of a single cell without cell disruption. A novel visible reporter was developed, designed for targeting to the S. cerevisiae cell surface, using a green fluorescent protein (GFP) from the jellyfish, Aequorea victoria (Chalfie et al., 1994). The fluorescence of wild-type GFP originates from a fluorescent chromophore (p-hydroxybenzylidene-imidazolidinone), formed upon cyclization by post-translational modification of the residues Ser, dehydro-Tyr and Gly within the hexapeptide, starting from the amino acid residue 64 in the 238-residue polypeptide (Cubitt et al., 1995). Formation of the fluorescent chromophore can occur in the absence of any cofactor, except for molecular oxygen. The chromophore absorbs blue or UV light with a maximum excitation at 395 nm (a minor band at 470 nm, and maximum excitation at 488 nm in the case of the enhanced GFP), and emits maximum fluorescence at 510 nm (with a shoulder at 540 nm) (Terry et al., 1995). The conformation of GFP is extremely stable with apparently little freedom and is degraded only under harsh environmental conditions, such as low pH or exposure to chaotropic ions, etc. The denatured GFP can often be refolded by renaturation (Ward and Bokman, 1982). GFP has been exploited as an extremely useful intracellular marker for gene expression and protein localization (Chalfie et al., 1994; Cubitt et al., 1995; Terry et al., 1995; Ward and Bokman, 1982; Brendan et al., 1997; Ram et al., 1998). To target GFP to the cell surface of S. cerevisiae and exploit it as a visible reporter, GFP was fused to the C-terminal half of ␣-agglutinin. The fused gene was further combined

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with the gene for a protein secretion signal. A promoter that has a positive response to carbon metabolism was employed to control the expression of the GFP-fusion protein as a model (Fig. 9). A glucose-inducible promoter GAPDH was employed as a model promoter to control the expression of the reporter gene in response to environmental changes. This designed gene was integrated into the chromosome of S. cerevisiae by homologous recombination. Fluorescence microscopy demonstrated that the transformed cells emitted green fluorescence, derived from the functionally expressed GFP contained in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody against GFP as the first antibody and Rhodamine Red TM-X-conjugated goat anti-rabbit IgG as the second antibody (impenetrate to the cell membrane). Furthermore, the display of GFP on the cell surface was confirmed using confocal laser scanning microscopy and by measuring fluorescence in each cell fraction, obtained after subcellular fractionation. As GFP was proved to be displayed in active form on the cell surface, selection of promoters will endow yeast cells with the ability to respond to the changes of environmental conditions, including nutrient concentrations in the media, through emission of fluorescence. The cell surface reporter system developed will have much more powerful capacity to detect molecular events inside or outside living cells than the original system in which the reporters were expressed in the cells. So far various kinds of promoters, which have either positive or negative response to the intracellular molecular events, have been identified and characterized. Most of them could be used to control the display of GFP on the cell surface, which allows us to measure the intracellular concentration of metabolites in vivo as the activators or repressors onto the promoters. A variety of GFP-type fluorescent proteins could also be used in this system to give different colors responding to each molecular event occur-

Fig. 9. Arming yeast with emission of GFP from the cell surface of yeast in response to the environmental conditions.

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ring inside and outside of the cells. It should be pointed out that, at present, it is not difficult to measure the extracellular concentrations of metabolites or nutrients on-line with current techniques. Nevertheless, on-line and in vivo detection of intracellular concentrations of metabolites at the single-cell level is still not available (Ye et al., in press). 5. Strategies for protein-immobilization on the yeast cell surface — construction of arming cells Surface-expression systems were initially reported by showing that peptides could be fused to the docking proteins (pIII) of a filamentous phage, without affecting its ability to infect Escherichia coli (Scott and Smith, 1990). This has led to the development of phage-display systems (Chiswell and McCafferty, 1992), and the isolation of specific ligands, antigens, and antibodies from complex libraries (Hoogenboom, 1997) have been carried out. However, hybrids of larger-sized polypeptides with the major coat protein of phage were not readily incorporated into the phage particles. The bacterial surface may be a more suitable tool for displaying higher numbers of proteins. On the surface of Gram-negative bacteria, a number of heterologous proteins have been displayed on the cell surface (Little et al., 1993; Georgiou et al., 1993, 1997), fused to surface-exposed termini of outer membrane proteins (Francisco et al., 1992, 1993). Lipoproteins (Harrison et al., 1990), fimbriae (Hedegaard and Klemm, 1989) and flagellar proteins (Newton, 1989) have also been used to display heterologous proteins on the cell surface. Gram-positive bacteria have also been taken into consideration for cell-surface display of heterologous proteins, for example, by using protein A localized on the cell wall (Gunneriusson et al., 1996; Samuelson et al., 1995; Schneewind et al., 1995). However, when cell-surface display is intended to be applied to bioindustrial processes for foods, alcoholic beverages, medicines, and so on, the most suitable microorganism is the yeast S. cerevisiae. This yeast benefits from ‘GRAS’ (generally regarded as safe) status and can be used for food and pharmaceutical production. Thus, S. cerevisiae is a particularly useful organism for developing a cell-surface expression system. It is also an advantageous host cell system for genetic engineering, since it enables folding and glycosylation of expressed heterologous eukaryotic proteins and is easy to handle with ample genetic techniques available. Moreover, yeast can be cultivated to a high cell density in relatively inexpensive media. The yeast S. cerevisiae is commonly used in the brewing industry and in the commercial fermentation of carbohydrates to ethanol. S. cerevisiae lacks both amylolytic and cellulolytic activities and is unable to ferment starchy or cellulosic materials, although they are the most abundant and utilizable resources of plant origin (Barnett, 1976). The immobilization of amylolytic and cellulolytic enzymes on the yeast cell surface may thus facilitate the utilization of starch and cellulose by yeast. In this system, the cell surface of yeast has been used as a carrier for immobilization of enzymes, and the living whole cells were remade as a ‘cell biocatalyst’. These surface-engineered yeast cells were named ‘Arming yeast’ (Anonymous, 1997). Although various methods for immobilization of enzymes have been developed to construct bioreactors with effective conversion of substrates, it is still difficult to maintain enzyme activities over the long reaction period or to perform multi-step conversions. The displayed enzymes can be regarded as a kind of self-immobilized enzyme system on the cell

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Fig. 10. Arming yeast constructed by cell surface engineering and the development of its application in biotechnology.

surface, this feature being passed on to daughter cells as long as the genes are retained by the cells. This display system could convert S. cerevisiae into a novel and attractive microorganism as a whole-cell biocatalyst by surface expression of various enzymes, especially when target substrates are not able to be taken up by the cells. The approach will make it possible to produce renewable self-immobilized biocatalysts. Furthermore, combination of the system with the visible reporter molecule may enable wide application in bioprocess engineering, such as on-line or in vivo monitoring of cell growth and metabolism at the single-cell level, since the fluorescence of GFP can be easily detected due to its display on the cell surface. Development of a visible reporter on the cell surface is designed to provide a quantitative evaluation system for cell surface engineering. The in vivo quantification of enzyme activity or of the total amounts of proteins displayed on the cell surface is not an easy task. Although a fluorescent antibody method may be used for this purpose, antibody can only be bound with the outer-layer proteins displayed on the cell surface. The proteins localized in the inner-layer may not have a chance to bind with the antibody. This renders quantitative assay very difficult. The display of a visible reporter on the cell surface may provide a solution to this problem, because it can be directly measured using confocal laser scanning microscopy combined with appropriate image processing. Fluorescence can also be visualized using a flow cytometer or a fluorescence-activated cells sorting apparatus. This is an important advantage for characterizing cells at the single cell level in a mixed cell population.

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We believe that the cell surface engineering described in this review will allow us to endow all living cells with novel abilities (Fig. 10) and exploit a new field in biotechnology.

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