Accepted Article

Received Date : 09-May-2014 Revised Date

: 14-Sep-2014

Accepted Date : 15-Sep-2014 Article type Editor

: MiniReview : Verena Siewers

Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology

Tsutomu Tanaka and Akihiko Kondo*

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan. *Corresponding author: Akihiko Kondo Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho Nada, Kobe 657-8501, Japan. Tel/Fax: +81-78-803-6196 E-mail: [email protected]

Running title: Yeast cell-surface display for synthetic biology Keywords: cell surface display, yeast, synthetic biology, metabolic engineering, protein secretion Abstract In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1567-1364.12212 This article is protected by copyright. All rights reserved.

Accepted Article

utilized as a cell factory for the production of fuels, chemicals and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. Introduction Cell-surface display techniques allow for the expression of a target peptide or protein on the surface of a cell through linkage with a genetically fused anchor protein. Microbial cell-surface display systems have numerous potential applications, including vaccine and antibody development, library screening, bioconversion, and biosorption (Lee et al., 2003; Lofblom et al., 2011). In particular, such tequniques have attracted attention for improving the biomass-degrading activities of yeast by displaying cellulases on the cell surface, thereby enabling direct ethanol production from biomass such as cellulose and starch (reviewed in Tanaka et al., 2012). Metabolic engineering is an integrated, multidisciplinary approach for optimizing a desired cellular property or phenotype, mainly engineering metabolic pathways and their regulation (Liu et al., 2007; Tyo et al., 2007; Hong et al., 2012; Nielsen et al., 2013). The field of metabolic engineering is rapidly evolving due to advances in cell engineering tools such as genetic manipulation, foundational technologies for functional genomics, computational systems biology, “omics” technologies, systems-level models of function, and an expanding repository of publicly available whole-genome sequences. These tools have directly contributed to the engineering of various industrially useful microbial strains (Curran & Alper, 2012). The approaches used to improve the productivity of native products, particularly ethanol, from metabolically engineered yeast have also been applied to industrially useful chemicals, fuels, pharmaceuticals, polymers, and proteins. Concurrently, the field of synthetic biology has helped accelerate the understanding of biological whole/local systems and the design, construction, and creation of cell factories. Synthetic biology and metabolic engineering are synergistically related fields, but use fundamentally different approaches. Metabolic engineering typically utilizes a top-down approach to optimize and reengineer metabolic networks for the overproduction of target compounds, whereas synthetic biology tends to proceed from a bottom-up approach for the construction of new biological systems. Advances in both fields have increased the knowledge of biological systems and desired synthetic pathways, which is essential for the development of novel cell factories (Nielsen et al., 2014). This article is protected by copyright. All rights reserved.

Accepted Article

Yeast cell-surface display techniques are dependent of the efficient expression and secretion of target proteins. Many engineering approaches have been used to improve and optimize the production of proteins by S. cerevisiae, including optimizing the fermentation process, selecting the most appropriate expression vectors and signal sequences for extracellular targeting, and engineering host strains with enhanced folding and post-translational modification systems (Idiris et al., 2010). Using these approaches, heterologous protein production has been improved from milligrams to grams per liter. However, improved productivity has only been achieved for a few proteins, and the development of a general cell factory platform for protein production remains difficult. Increasing protein productivity by yeast is challenging due to the complexity of protein processing, post-translational modification, and secretion pathways, which involve protein folding, glycosylation, disulfide bond formation, and vesicle trafficking (Idiris et al., 2010). By increasing our understanding of both the individual processes and the overall system through integrated analysis, general models for protein secretion can be derived and utilized for engineering efficient secretory pathways and cell factories for recombinant protein production (Graf et al., 2009). Systems biology approaches are expected to substantially enhance cell-surface display, both in terms of providing system-level understanding and for identifying engineering targets (reviewed in Hou et al., 2012). Many studies on partially engineering protein secretion pathway could improve protein secretion ability, and Omics analysis is helpful to understand cellular behavior and to plot potential strategies for protein secretion. Even though there are some examples of systems biology based approach for increasing protein secretion (Kroukamp et al., 2013; Van Zyl et al., 2014), improvement of surface display efficiency has not been demonstrated yet. The present review focuses on recent advances in the field of cell-surface display, particularly approaches aimed at increasing the efficiency of display, which includes the amount and activity/function of the target protein displayed on the cell surface. The other approaches constructing cellulosome on the surface by synthetic biology are also summarized. Most cell factories, including yeast, are capable of producing the desired product using glucose as a carbon source. However, to improve the cost efficiency of microbial biosynthesis, altering the carbon flux toward the product of interest is required for minimizing the amount of carbon lost to byproducts and cellular growth. To this end, cell-surface display can increase the variety of available sugars, such as cellobiose and cellooligosaccharides, from inexpensive starting materials by displaying cellulase on the yeast cell surface. In combination with metabolic engineering approaches and synthetic biology approaches, cell-surface display is required to construct engineered yeast cells with a desired function (Figure 1). As few systems biology studies on metabolic networks involving cell-surface display have been reported, here, important considerations for designing and re-constructing This article is protected by copyright. All rights reserved.

Accepted Article

cell-surface display systems, including protein expression, secretion, and anchoring to the cell surface are presented. Selecting an appropriate anchor protein: N- or C-terminal fusions N-terminal fused anchor proteins Selecting an anchor protein is an important consideration for cell-surface display systems (Tanaka et al. 2012). An ideal anchor protein contains a signal sequence that mediates its efficient transport to the cell surface, where it strongly immobilizes the fused target protein, and does not interfere with the stability or activity of the displayed protein. The choice of the anchor protein is thus dependent upon the specific application and properties of the target protein. For N-terminal fusions, the N-terminus of the anchor protein is genetically fused to the C-terminus of the target protein. A signal sequence for secretion is also fused to the N terminus of the target protein to facilitate transport to the cell surface. The resultant fusion protein is then expressed by the host, such as the yeast S. cerevisiae (Figure 2). Alpha-agglutinin was one of the commonly utilized anchor protein for N-terminal fusion. Alpha-agglutinin has four regions, a secretion-signal region, active region, serine- and threonine-rich support region, and a putative glycosylphosphatidylinositol (GPI) anchor-attachment signal. Several different C-terminal fragments of α-agglutinin (i.e. different molecular sizes) can be fused with the target protein, but all contain the GPI anchor motif. In addition to α-agglutinin, Van der Vaart et al. (1997) demonstrated that a number of cell wall proteins of S. cerevisiae (Cwp1p, Cwp2p, Agα1p, Tip1p, Flo1p, Sed1p, YCR89w, and Tir1) can be used as N-terminally fused anchors. The choice of anchor protein is one of the important factor for the total amount of protein displayed and expressed, and it can enhance the accessibility of a substrate to a displayed enzyme. Increasing the length of the anchor protein can increase access to the cell-surface displayed protein. For example, both the accessibility and enzymatic activity of cell-surface displayed gluco-amylase increased with increasing molecular size of the anchor protein Flo1p (Sato et al., 2002). Length between the target protein and anchor proteins will affect target protein accessibility. Breinig & Schmitt (2002) showed that insertion of a 350-residue Ser/Thr-rich spacer between hemagglutinin peptide (HA peptide) and either Cwp2 or Flo1p used as an anchor dramatically increases accessibility to hemagglutinin on the yeast cell surface. Washida et al. (2001) evaluated changes in the length of the spacer between Rhizopus oryzae lipase and α-agglutinin and demonstrated that the spacer is required to separate the active enzyme from the anchor. Interestingly, the authors also found that the enzymatic activity depended on length of the spacer, suggesting optimization of the spacer length could improve the function of displayed target proteins used as whole-cell biocatalysts (Washida et This article is protected by copyright. All rights reserved.

Accepted Article

al., 2001; Tanaka et al., 2012). C-terminal fused anchor proteins The fusion site between target and anchor proteins may significantly impact whether the target protein retains its function when displayed (Matsumoto et al., 2002). For example, enzymes in which the active site is located near the C-terminus, such as lipases and glycosyltransferases, are not suitable for N-terminal display systems because positioning the GPI anchor sequence at the C terminus can impair the catalytic activity of the enzyme. In C-terminal fusion systems, the C-terminus of the anchor protein is genetically fused to the N-terminus of the target protein (Figure 2). The most widely used anchor for C-terminal fusions is Aga2p. The cell wall protein a-agglutinin consists of two subunits, Aga1p and Aga2p. Aga1p is incorporated in the cell wall through a GPI anchor, whereas Aga2p is anchored to the cell wall Aga1p via two disulfide bonds to Aga1p. Boder & Wittrup (1997) developed a sophisticated approach to combinatorial library screening using an Aga2p display platform. Pir proteins are also used as anchors in C-terminal fusion cell-surface display systems. Pir proteins covalently bind to β-1,3-glucan in the yeast cell wall, despite the absence of a C-terminal GPI-anchoring signal. This binding occurs through an unknown linkage that is sensitive to mild alkaline treatment (Ecker et al., 2006). Yeast cells displaying glycosyltransferases anchored to Pir proteins were shown to synthesize the target oligosaccharide (Abe et al., 2003). Truncated forms of Flo1p (FL and FS, corresponding to residues 1-1447 and 1-1099, respectively) are also used as C-terminal anchors (Matsumoto et al., 2002). C-terminal fusion methods using FL and FS as anchors exploit the adhesive character of the flocculation functional domain of Flo1p, which apparently binds to cell surface mannan chains via noncovalent interactions.

Improvement of yeast cell-surface display efficiency Traditional approaches to improve protein secretion and display efficiency Studies investigating methods for improving protein secretion are expected to be directly applicable for increasing the display efficiency of target proteins on the yeast cell surface. Detailed knowledge of the yeast secretion pathway has enabled secretion efficiency and protein yields to be improved through a combination of various molecular techniques for the optimization of signal sequences, upreguration of ER folding, modification of vesicle transport, and deletion of host proteinases (Idiris et al., 2011; den Haan et al., 2013). Systems biology tools for the evaluation of such engineered strains will help identify novel targets for further improving protein secretion (reviewed in Hou et al., 2012), as well as protein display This article is protected by copyright. All rights reserved.

Accepted Article

efficiency. Signal sequences are key factors controlling protein secretion. Native S. cerevisiae leader sequences, in addition to foreign and synthetic leader sequences, have been used to target heterologous proteins for secretion. Signal sequences that are able to enhance protein secretion have also been successfully engineered (Hou et al., 2012). In the case of cell-surface display systems, a foreign signal sequence derived from Rhizopus oryzae has been used in conjunction with α-aggulutinin as an anchor protein, and a native Aga2 signal sequence was used for Aga2p anchor protein (Tanaka et al., 2012). However, strategies for engineering signal sequences are needed to further improve target protein display efficiency (Rakestraw et al., 2009). With respect to enhancing protein secretion, promoting glycosylation reactions in the ER and the Golgi by modifying the amino acid sequence of the target protein or glycosylation enzymes may be a promising approach. Although the effect of glycosylation on protein secretion is not fully understood, glycosylation has been shown to reduce protein aggregation (Parthasarathy et al., 2006) and degradation (Rudd et al., 2004). Disulfide bonds are another factor that affect protein secretion, and the over-expression of chaperones such as Kar2p and PDI can increase target protein secretion (Hou et al., 2012). Localization of GPI-anchored proteins on the yeast cell surface occurs through the exocytosis of proteins within secretory vesicles. Protein secretion in S. cerevisiae involves the transfer of targeted proteins through various membrane-enclosed compartments. Proteins targeted for secretion are first translocated into the lumen of the endoplasmic reticulum and are subsequently translocated to the Golgi apparatus, where they are transported to the plasma membrane in membrane-enclosed vesicles (Schekman, 1992). The fusion of Golgi-derived secretory vesicles with the plasma membrane releases the enclosed proteins outside of the cytosol. Prior to their secretion, post-translational proteolytic modification of secretory precursor proteins occurs in the trans cisternae of the Golgi apparatus and secretory vesicles during the late secretory pathway. In S. cerevisiae, the Kex2 endopeptidase is situated in the trans cisternae of the Golgi apparatus to eliminate the proregion of precursor proteins, such as the α-factor pheromone used in mating. α-Agglutinin is thought to be transported by secretory vesicles in a GPI-anchored form to the exterior of the plasma membrane, where it is released by phosphatidylinositol-specific phospholipase C and then transferred to the cell wall. The anchoring of α-agglutinin in the outermost surface of the cell wall is accomplished by the addition of β-1,6-glucan to the GPI anchor remnant (Lu et al., 1994; Lu et al., 1995; Kapteyn et al., 1996; Shibasaki et al., 2009). The amount of expressed proteins on the yeast cell surface was evaluated using EGFP as a reporter (Shibasaki et al., 2001). The fluorometric and image analysis showed 104–105 molecules of α-agglutinin-fused proteins per cell were expressed on the cell surface. In the case of Pichia pastoris, 104 molecules of (Candida antarctica lipase B) CALB This article is protected by copyright. All rights reserved.

Accepted Article

molecules were immobilized on the single cell surface using FS anchor system (Liang et al., 2013). Semi-rational approaches to improve cell-surface display efficiency The most apparent semi-rational approach for enhancing the display efficiency of yeast is the deletion or overexpression of genes of host cells. The display efficiency of a SED1-disrupted yeast strain was evaluated using monomeric red fluorescent protein as a marker (Kuroda et al., 2009). Sed1p is a major cell wall structural protein that is primarily expressed in the stationary phase, but is also induced by stress and starvation (Shimoi et al., 2008). Similar to α-agglutinin, Sed1p has a GPI anchor attachment signal at the C-terminus and is incorporated into the cell wall via a β-1,6-glucan side chain. SED1 disruption is considered to reduce the competition between α-agglutinin-fused proteins and Sed1p for cell surface expression during the stationary phase. The monomeric red fluorescent protein derived fluorescence intensity of the SED1-disrupted yeast strain after 72h cultivation was 1.47-fold higher than that of the wild-type strain (Kuroda et al., 2009). Environmental substances have limited contact with the plasma membrane due to the presence of a bulky layer of glucan and mannose polymers in the cell wall, and the disturbance and loosening of the mannoprotein network are considered to increase cell wall porosity (Zlotnik et al., 1984). Consequently, cell-surface displayed proteins in SED1-disrupted cells are considered to be more accessible to substrate molecules, possibly through the outer localization or due to the lack of steric hindrance by cell wall polymers. Using a high-efficiency loss of heterozygosity (HELOH) method, a diploid sake yeast strain with deleted SED1 gene was constructed and shown to have 1.6-fold higher activity of cell-surface displayed BGL compared to that of the wild-type strain (Kotaka et al., 2010). Potential candidate gene(s) for deletion to improve cell-surface display efficiency were identified using a cell surface engineering approach (Matsuoka et al., 2014). Screening for cell wall mutants with cell surface environments suitable for protein display revealed that deletion of Mnn2 increased BGL activity on the yeast cell surface by approximately 1.6 fold compared to that of wild-type strain. In addition, endo-glucanase activity on the cell surface of the Mnn2-deleted strain was also increased up to 1.9-fold. The amount of the displayed target protein on the Mnn2-deleted yeast strain cell surface was increased, and the mannan level was decreased, suggesting that the cell surface environment of the Mnn2-deleted strain facilitates the binding of high-molecular-weight substrates to the active sites of cell-surface displayed enzymes. Combinatorial approach to enhance cell-surface display efficiency The overexpression of proteins involved in cell-surface display is another approach to improve display efficiency. Wentz et al. (2007) attempted to identify genes that enhance This article is protected by copyright. All rights reserved.

Accepted Article

protein secretion by screening for gene products that improve surface display efficiency. In their approach, cDNA overexpression libraries were used to generate target-protein displaying yeast. Prior to introduction of the plasmid-based cDNA library, yeast cells were transformed with a plasmid encoding candidate protein and its expression was then induced in galactose-based medium. The yeast cells were then probed for active surface-tethered proteins and analyzed by flow cytometry. The display-enhancing genes were identified and the phenotype of each strain was also characterized. Although one of the candidate proteins was particularly sensitive to the growth conditions, several yeast strains that promoted increased display levels and secretion efficiency were isolated. The identified genes were RPP0 (overexpression of translational components), ERO1 (ER-resident folding assistant) and the cell-wall related proteins SED1, CCW12, and CWP2. CCW12 and ERO1 are the most generalizable enhancers of target protein secretion; notably, ERO1 increases the secretion of active protein by up to 7.9-fold. However, the secretion levels were dependent on the protein of interest, suggesting that the display efficiency is also dependent on the characteristics of the target and anchor proteins. Optimization of cultivation conditions Cultivation conditions are also a key determinant of cell-surface display efficiency. In the case of Aga2-based yeast display systems, the cells are typically grown initially in glucose- or raffinose- containing medium to an optical density at 600 nm of between 2–5, and galactose-additional medium is then used for an additional 48–72 h of cultivation (Puthenveetil et al., 2009; Chen et al., 2011; Han et al., 2011; Lee et al., 2011; Dang et al., 2012, Blazic et al., 2013) However, Andreu & Del Olmo (2013) demonstrated that a long incubation period with galactose (48-72 h) is not required to achieve good display efficiency. Although longer cultivation typically results in a larger amount of produced proteins, plasmid loss also increases; therefore, 6–8 h of incubation is suitable for good levels of display efficiency. Blazic et al. (2013) reported that a 12h incubation in galactose was appropriate for optimizing display efficiency. These reports also show that cell-surface display efficiency is dependent on the anchor and target protein and peptides. Ethanol production using cellulase- or amylase-displaying yeast strains Lignocellulosic materials are an attractive, environmentally friendly feedstock since sustainable biomass can be converted into bio-based chemicals or liquid fuels such as bioethanol, which is currently one of the most promising alternatives to conventional fossil fuels (Elkins et al., 2010; Peralta-Yahya et al., 2012). Lignocellulosic materials, including grass, wood, and their residues, are predominantly composed of cellulose, hemicellulose and lignin. To harness these materials for bioconversion processes using microorganisms, they must be degraded into compounds that can be used as nutrients by microorganisms. However, This article is protected by copyright. All rights reserved.

Accepted Article

the key factor limiting the industrial production of cellulosic bioethanol is the slow rate of microbial degradation of cellulose into fermentable sugars (Himmel et al., 2007). Although cellulosic biomass is synergistically hydrolyzed by cellulases during the saccharification and fermentation steps, the process consumes large amounts of cellulase enzymes and is expensive and time-consuming. Consolidated bioprocessing (CBP) that combines enzyme production, cellulose hydrolysis, and fermentation into a single process is reported to significantly reduce the cost of cellulosic ethanol production (Lynd et al., 2002; Lynd et al., 2005). Cellulase-displaying S. cerevisiae strains have been used as whole-cell biocatalysts for bioethanol production by CBP. Several groups have also produced ethanol from starchy materials using α-amylase- and glucoamylase-displaying yeast strains (Shigechi et al., 2004; Seong et al., 2006; Kotaka et al., 2008a, b; Yamakawa et al., 2010; Yamada et al., 2011). Several different glucoamylases can be displayed on the yeast cell surface in fully active form using α-agglutinin as an anchor. The co-display of α-amylase and glucoamylase has also been demonstrated (Shigechi et al., 2004), and amylase-displaying yeast can be used in repeated batch cultures (Yamakawa et al., 2010), because displayed enzyme was easily recovered along with cells by centrifugation. An amylase-displaying yeast strain has been used for direct ethanol production from natural biomass, such as high-yield rice (Yamada et al., 2011). Cellulose is the most abundant biomass and is a promising renewable carbon source. However,. many enzymes, including endoglucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (BGL), are required for cellulose degradation (den Haan et al., 2013) because most microbes have difficulty assimilating cellulose due to its rigid structure (Elkins et al., 2010). Co-display of these enzymes on the yeast cell surface promotes the direct fermentation of ethanol from cellulosic materials or production of ethanol from β-glucan (Fujita et al., 2002; Kotaka et al., 2008a, b) or amorphous cellulose (Fujita et al., 2004). In addition, Katahira et al. (2004, 2006) demonstrated the direct fermentation of xylan by a yeast strain co-displaying xylanase and β-xylosidase. Nakamura et al. (2008) also reported the efficient co-fermentation of xylose and cellobiose using a β-glucosidase-displaying yeast strain, which is able to convert cellobiose to glucose on the cell surface and then utilize the glucose for growth. This system enables the extracellular glucose concentration to be controlled, thereby avoiding catabolite repression. Reconstruction of cellulosomes on the yeast cell surface Construction of cellulolytic enzyme networks on the cell surface by synthetic approaches has been demonstrated recently. Cellulosomes are multi-enzyme complexes produced by anaerobic bacteria for the efficient deconstruction of cellulose and hemicellulose (Bayer et al., 2004). Due to a highly specific interaction between the enzyme-bearing dockerin and resident cohesin modules of the scaffoldin, the enzymatic subunits of the cellulosome complex can be This article is protected by copyright. All rights reserved.

Accepted Article

assembled onto the cell surface, which allows their catalytic activities to work synergistically. Compared with non-complexed cellulase systems, cellulosomes are assembled by noncatalytic scaffoldin protein through high-affinity interactions between scaffoldincohesins and enzyme-borne dockerins (Lynd et al., 2002). Scaffoldin typically contains a cellulose-binding domain that can anchor the entire complex onto the cellulosic substrate (Doi & Kosugi 2004). The assembly of cellulases onto the scaffoldin leads to a synergistic relationship between the enzymes that is further augmented by the enzyme–substrate targeting through the cellulose-binding domain (Bayer et al., 2004). Cellulosomes can also reduce the diffusion of substrate and products, allowing for the efficient uptake of oligosaccharides by the host (Bayer et al., 1994; Schwarz, 2001). Major progress has been accomplished in the production of mini-cellulosomes and artificial cellulosomes that efficiently degrade crystalline cellulose (Kataeva et al., 1997; Fierobe et al., 2001; Fierobe et al., 2002; Murashima et al., 2002; Fierobe et al., 2005; Mingardon et al., 2007). A recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes was first reported by Zhao’s group (Wen et al., 2010). These minicellulosomes consisted of a miniscaffoldin, which contained a cellulose-binding domain and three cohesin modules and was tethered to the cell surface through the yeast α-agglutinin anchor, and three types of cellulases (EG, CBH, and BGL), each bearing a C-terminal dockerin. Two types of surface-displaying strains were constructed (Figure 1): one displayed a trifunctional minicellulosome with EGII, CBHII, and BGL1 activities, and the other displayed three types of unifunctional minicellulosomes: one with EGII activity, one with CBHII activity, and one with BGL1 activity. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme and enzyme proximity synergies, which contributed to the final ethanol yields and productivity of 1.8 g ethanol/liter and 0.31 g ethanol produced per g of phosphoric acid swollen cellulose consumed, respectively. Because the yeast surface-displayed unifunctional minicellulosomes were not capable of two-dimensional diffusion, they were spatially distributed on the yeast cell surface. The yield and ethanol productivity from phosphoric acid swollen cellulose were not sufficiently high for practical applications, a result that may have been due to the saturation of the secretion pathway. To improve cellulose degradation ability, Chen’s group constructed a synthetic yeast consortium using four kinds of minicellulosome-displaying yeast strains (Tsai et al., 2010). The dockerin-cohesin pairs were obtained from three different microorganisms and each pair had specific interactions, permitting the highly controllable ordering of each enzyme in the minicellulosome complex. By adjusting the ratio of the different populations in the consortium, the cellulosome assembly on the yeast cell surface, cellulose hydrolysis, and ethanol production can be easily optimized. The synthetic consortium after optimization produced nearly twice the level of ethanol as the consortium This article is protected by copyright. All rights reserved.

Accepted Article

with an equal proportion of the different yeast populations. Simultaneous cell growth and ethanol production from phosphoric acid swollen cellulose was next achieved using α-agglutinin as an anchor and an optimized expression system (Goyal et al., 2011). Using this approach, ethanol production was increased by approximately three fold. A tetravalent cellulosome was constructed using a natural adaptive assembly strategy (Tsai et al., 2013). Yeast cells expressing the tetravalent celluosome on the surface exhibited a two-fold increase in ethanol production compared with cells displaying a divalent cellulosome at a similar enzyme loading level (Tsai et al., 2013). These results suggest that enzyme proximity is more important for cellulosomal synergy than the amount of enzyme loaded on the cell surface. A modified cellulosome consortium was designed for the cell-surface display of EG and CBH via a cohesion-dockerin interaction, in addition to BGL, which imparted a cell wall adhesion property and was expressed without a dockerin (Kim et al., 2013). The optimized ratio of high ethanol productivity was easily achieved by simply changing the combination ratio of the different yeast populations. Another approach for constructing an artificial cellulosome utilized two types of cohesion-dockerin interactions (Fan et al., 2012). A type I cohesin–dockerin interaction was introduced to construct minicellulosomes, whereas a type II cohesin–dockerin interaction was used to mediate anchoring of the cellulosomes onto the cell surface. The species specificity of these two interactions ensures that the cellulosome assembly and cell surface attachment steps are distinct and also allows for control of the grafting of catalytic units. The construction of a minihemicellulosome and xylanosome for hemicellulose hydrolysis has also been reported (Sun et al., 2012; Srikrishnan et al., 2013). Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, and is a principal component of plant cell walls. The minihemicellulosome was comprised of an endoxylanase, arabinofuranosidase, and β-xylosidase, with each enzyme bearing a C-terminal dockerin. With an integrated D-xylose-utilizing pathway, the recombinant yeast displaying the minihemicellulosome complex on the cell surface simultaneously hydrolyzed and fermented birchwood xylan to ethanol with a yield of 0.31 g ethanol per g of sugar consumed. (Sun et al., 2012)

Conclusion Cell-surface display is a powerful tool for functionalizing yeasts to serve as whole-cell biocatalysts and/or platforms for the study of proteins. A novel intein-based cell-surface display system has been developed (Marshall et al., 2013) and using other cell surface display techniques, several proteins, including xylose isomerase (Ota et al., 2013), laccase (Nakanishi et al., 2012), and expansin-like protein (Yamada et al., 2013) have been This article is protected by copyright. All rights reserved.

Accepted Article

displayed on the yeast cell surface. Although numerous recent applications for surface display systems have been reported (Wilde et al., 2012; Fushimi et al., 2013; Wang et al., 2013; Yi et al., 2013; Zhang et al., 2013; Qiu et al., 2014), the identification of a wider variety of anchor proteins is a promising way to broaden the potential applications of yeast cell-surface display techniques. Systems biology approaches, such as cell surface analysis, omics technologies, redesign of secretory pathways, and strain engineering, will enable faster progress in the development of cell-surface display techniques. Many of the systems-levels approaches and mathematical models for optimization of cellular metabolic pathway have been based on the direct utilization of monomeric sugars such as glucose or xylose as carbon sources. Synthetic biology has extended the spectrum of produced biofuels/biochemicals by introducing novel metabolic pathways, as well as increasing their productivity. Alternatively, cellulase-displaying yeast strains are able to utilize oligomeric sugars as carbon sources and to keep the concentration of extracellular monomeric sugars at low levels. Hence, the combination of synthetic and systems biology approaches with cell-surface display technology has the potential to create novel microbial factories that are capable of utilizing oligomeric sugars and cellulose as carbon sources for the production of industrially important compounds.

Acknowledgements This work was mainly supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan, and by Grant-in-Aid for Scientific Research (B) (Gran number: 24310065), MEXT, Japan.

References Abe H, Shimma Y & Jigami Y (2003) In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein. Pir Glycobiology 13: 87–95. Andreu C & Del Olmo M (2013) Yeast arming by the Aga2p system: effect of growth conditions in galactose on the efficiency of the display and influence of expressing leucine-containing peptides. Appl Microbiol Biotechnol 97: 9055-9069. Bayer EA, Belaich JP, Shoham Y & Lamed R (2004) The cellulosomes: Multienzyme machines for degradation of plant cell wall polysaccharides. Annu Rev Microbiol 58: This article is protected by copyright. All rights reserved.

Accepted Article

521–554. Bayer EA, Morag E & Lamed R (1994) The cellulosome—a treasure-trove for biotechnology. Trends Biotechnol 12:379–386. Blazic M, Kovacevic G, Prodanovic O, Ostafe R, Gavrovic-Jankulovic M, Fischer R & Prodanovic R (2013) Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases. Protein Expr Purif 89: 175–180. Boder ET & Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15: 553–557. Breinig F & Schmitt MJ (2002) Spacer-elongated cell wall fusion pro- teins improve cell surface expression in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 58: 637–644. Chen I, Dorr BM & Liu DR (2011) A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA 108: 11399–113404. Curran KA & Alper HS (2012) Expanding the chemical palate of cells by combining systems biology and metabolic engineering. Metab Eng 14: 289-297. Den Haan R, Kroukamp H, van Zyl JH, Willem H & van Zyl WH (2013) Cellobiohydrolase secretion by yeast: Current state and prospects for improvement. Process Biochem 48: 1-12. Doi RH & Kosugi A (2004) Cellulosomes: Plant-cell-wall-degrading enzyme complexes. Nat Rev Microbiol 2:541–551. Ecker M, Deutzmann R, Lehle L, Mrsa V & Tanner W (2006) Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage. J Biol Chem 281:11523-11529. Elkins JG, Raman B, Keller M (2010) Engineered microbial systems for enhanced conversion of lignocellulosic biomass. Curr Opin Biotechnol 21:657-662. Fan LH, Zhang ZJ, Yu XY, Xue YX & Tan TW (2012) Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production. This article is protected by copyright. All rights reserved.

Accepted Article

Proc Natl Acad Sci U S A 109: 13260-13265. Fierobe HP, Mechaly A, Tardif C, Belaich A, Lamed R, Shoham Y, Belaich JP & Bayer EA (2001) Design and production of active cellulosome chimeras: selective incorporation of dockerin-containing enzymes into defined functional complexes. J Biol Chem 276: 21257–21261. Fierobe HP, Mingardon F, Mechaly A, Bélaïch A, Rincon MT, Pagès S, Lamed R, Tardif C, Bélaïch JP & Bayer EA (2005) Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined tri-functional scaffoldin. J Biol Chem 280: 16325–16334. Fujita Y, Takahashi S, Ueda M, Tanaka A, Okada H, Morikawa Y, Kawaguchi T, Arai M, Fukuda H & Kondo A (2002) Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. Appl Environ Microbiol 68: 5136–5141. Fushimi T, Miura N, Shintani H, Tsunoda H, Kuroda K & Ueda M (2013) Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by yeast cell surface engineering. Appl Microbiol Biotechnol 97: 4003-4011. Gibson DG, Glass JI, Lartigue C et al. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329: 52–56. Goyal G, Tsai SL, Madan B, DaSilva NA & Chen W (2011) Simultaneous cell growth and ethanol production from cellulose by an engineered yeast consortium displaying a functional mini-cellulosome. Microb Cell Fact 10: 89–96. Graf A, Dragosits M, Gasser B & Mattanovich D (2009) Yeast systems biotechnology for the production of heterologous proteins. FEMS Yeast Res 9: 335–348. Han T, Sui J, Bennett AS, Liddington RC, Donis RO, Zhu Q & Marasco WA (2011) Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display. Biochem Biophys Res Commun 409: 253–259. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW & Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315: This article is protected by copyright. All rights reserved.

Accepted Article

804-807. Hong KK & Nielsen J (2012) Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell Mol Life Sci 69: 2671-2690. Hong K-K, Vongsangnak W, Vemuri GN & Nielsen J (2011) Unravelling evolutionarysstrategies of yeast for improving galactose utilization through integrated systems level analysis. Proc Natl Acad Sci USA 108: 12179–12184. Hou J, Tyo KE, Liu Z, Petranovic D & Nielsen J (2012) Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae. FEMS Yeast Res 12: 491-510. Idiris A, Tohda H, Kumagai H & Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86: 403–417. Kapteyn JC, Montijn RC, Vink E, de la Cruz J, Llobell A, Douwes JE, Shimoi H, Lipke PN & Klis FM (1996) Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology 6: 337-345. Kataeva I, Guglielmi G & Béguin P (1997) Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes. Biochem J 326: 617-24. Katahira S, Fujita Y, Mizuike A, Fukuda H & Kondo A (2004) Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells. Appl Environ Microbiol 70: 5407–5414. Kim IK, Roldão A, Siewers V & Nielsen J (2012) A systems-level approach for metabolic engineering of yeast cell factories. FEMS Yeast Res 12:228-248. Kim S, Baek SH, Lee K & Hahn JS (2013) Cellulosic ethanol production using a yeast consortium displaying a minicellulosome and β-glucosidase. Microb Cell Fact 12:14. Kotaka A, Bando H, Kaya M, Kato-Murai M, Kuroda K, Sahara H, Hata Y, Kondo A & Ueda M (2008a) Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase. J Biosci Bioeng 105: 622–627. Kotaka A, Sahara H, Hata Y, Abe Y, Kondo A, Kato-Murai M, Kuroda K & Ueda M (2008b) This article is protected by copyright. All rights reserved.

Accepted Article

Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases. Biosci Biotechnol Biochem 72: 1376–1379. Kotaka A, Sahara H, Kuroda K, Kondo A, Ueda M & Hata Y (2010) Enhancement of beta-glucosidase activity on the cell-surface of sake yeast by disruption of SED1. J Biosci Bioeng 109: 442–446. Kroukamp H, Den Haan R, Van Wyk N, Van Zyl WH (2013) Overexpression of native PSE1 and SOD1 in Saccharomyces cerevisiae improved heterologous cellulase secretion. Appl Ener 102:150-156. Kuroda K, Matsui K, Higuchi S, Kotaka A, Sahara H, Hata Y & Ueda M (2009) Enhancement of display efficiency in yeast display system by vector engineering and gene disruption. Appl Microbiol Biotechnol 82: 713-719. Lee G-Y, Jung J-H, Seo D-H, Hansin J, Ha S-J, Cha J, Kim Y-S & Park C-S (2011) Isomaltulose production via yeast surface display of sucrose isomerase from Enterobacter sp. FMB-1 on Saccharomyces cerevisiae. Biores Tech 9: 9179–9184. Lee SY, Choi JH & Xu Z (2003) Microbial cell-surface display. Trends Biotechnol 21: 45–52. Liang XX, Wang BB, Sun YF, Lin Y, Han SY, Zheng SP & Cui TB (2013) Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system. Biotechnol Lett 35: 367-374. Liu L, Redden H & Alper HS (2007) Frontiers of yeast metabolic engineering: diversifying beyond ethanol and Saccharomyces. Curr Opin Biotechnol 24: 1023-1030. Lofblom J (2011) Bacterial display in combinatorial protein engineering. Biotechnol J 6: 1115–1129. Lu CF, Kurjan J & Lipke PN (1994) A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol Cell Biol 14: 4825-4833. Lu CF, Montijn RC, Brown JL, Klis F, Kurjan J, Bussey H & Lipke PN (1995) Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol 128: 333-340.

This article is protected by copyright. All rights reserved.

Accepted Article

Lynd LR, van Zyl WH, McBride JE & Laser M (2005) Consolidated bioprocessing of cellulosic biomass: An update. Curr Opin Biotechnol 16: 577–583. Lynd LR, Weimer PJ, van Zyl WH & Pretorius IS (2002) Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol Mol Biol Rev 66: 506–577. Marshall CJ, Agarwal N, Kalia J, Grosskopf VA, McGrath NA, Abbott NL, Raines RT & Shusta EV (2013) Facile chemical functionalization of proteins through intein-linked yeast display. Bioconjug Chem 24: 1634-1644. Matsumoto T, Fukuda H, Ueda M, Tanaka A & Kondo A (2002) Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation functional domain. Appl Environ Microbiol 68:4517–4522. Matsuoka H, Hashimoto K, Saijo A, Takada Y, Kondo A, Ueda M, Ooshima H, Tachibana T & Azuma M (2014) Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae. Yeast 31: 67-76. Mingardon F, Chanal A, Tardif C, Bayer EA & Fierobe HP. 2007. Exploration of new geometries in cellulosome-like chimeras. Appl Environ Microbiol 73: 7138 –7149. Mrsa V, Seidl T, Gentzsch M & Tanner W (1997) Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. Yeast 13: 1145–1154. Murashima K, Chen CL, Kosugi A, Tamaru Y, Doi RH & Wong SL (2002) Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes. J Bacteriol 184: 76-81. Nakamura N, Yamada R, Katahira S, Tanaka T, Fukuda H & Kondo A (2008) Effective xylose/cellobiose co-fermentation and ethanol production by xylose-assimilating S. cerevisiae via expression of beta-glucosidase on its cell surface. Enz Microb Tech 43: 233–236. Nakanishi A, Bae JG, Fukai K, Tokumoto N, Kuroda K, Ogawa J, Nakatani M, Shimizu S & Ueda M (2012) Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation. Appl Microbiol Biotechnol 94: 939-948.

This article is protected by copyright. All rights reserved.

Accepted Article

Nielsen J & Jewett MC (2008) Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae. FEMS Yeast Res 8: 122–131. Nielsen J, Larsson C, van Maris A & Pronk J (2013) Metabolic engineering of yeast for production of fuels and chemicals. Curr Opin Biotechnol 24: 398-404. Nielsen J, Fussenegger M, Keasling J, Lee SY, Liao JC, Prather K & Palsson B (2014) Engineering synergy in biotechnology. Nat Chem Biol 10:319-322. Nevoigt E (2008) Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 72: 379–412. Ota M, Sakuragi H, Morisaka H, Kuroda K, Miyake H, Tamaru Y & Ueda M (2013) Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation. Biotechnol Prog 29: 346-351. Park JH, Lee SY, Kim TY & Kim HU (2008) Application of systems biology for bioprocess development. Trends Biotechnol 26: 404–412. Parthasarathy R, Subramanian S, Boder ET & Discher DE (2006) Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display. Biotechnol Bioeng 93: 159–168. Peralta-Yahya PP, Zhang F, del Cardayre SB and Keasling JD (2012) Microbial engineering for the production of advanced biofuels. Nature 488:320-328. Puthenveetil S, Liu DS, White KA, Thompson S & Ting AY (2009) Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase. J Am Chem Soc 131: 16430–16438. Qiu Z, Tan H, Zhou S & Cao L (2014) Surface display of a bifunctional glutathione synthetase on Saccharomyces cerevisiae for converting chicken feather hydrolysate into glutathione. Mol Biotechnol in press. (DOI 10.1007/s12033-014-9750-4) Rakestraw JA, Sazinsky SL, Piatesi A, Antipov E & Wittrup KD (2009) Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae. Biotechnol Bioeng 103:1192-1201.

This article is protected by copyright. All rights reserved.

Accepted Article

Rudd PM, Wormald MR & Dwek RA (2004) Sugar-mediated ligand–receptor interactions in the immune system. Trends Biotechnol 22: 524–530. Ruder WC, Lu T & Collins JJ (2011) Synthetic biology moving into the clinic. Science 333: 1248–1252. Sato N, Matsumoto T, Ueda M, Tanaka A, Fukuda H & Kondo A (2002) Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates. Appl Microbiol Biotechnol 60: 469–474. Schekman R (1992) Genetic and biochemical analysis of vesicular traffic in yeast. Curr Opin Cell Biol 4: 587-592. Schekman R & Novick P (1982) The Molecular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 361. Schwarz WH (2001) The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 56:634–649. Seong KT, Katakura Y, Ninomiya K, Bito Y, Katahira S, Kondo A, Ueda M & Shioya S (2006) Effect of flocculation on performance of arming yeast in direct ethanol fermentation. Appl Microbiol Biotechnol 73: 60–66. Shibasaki S, Maeda H & Ueda M (2009) Molecular display technology using yeast--arming technology. Anal Sci 25: 41-49. Shibasaki S, Ueda M, Iizuka T, Hirayama M, Ikeda Y, Kamasawa N, Osumi M & Tanaka A (2001) Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses. Appl Microbiol Biotechnol 55:471–475. Shigechi H, Koh J, Fujita Y, Matsumoto T, Bito Y, Ueda M, Satoh E, Fukuda H & Kondo A (2004) Direct production of ethanol from raw corn starch via fermentation by use of a novel surface- engineered yeast strain codisplaying glucoamylase and alpha- amylase. Appl Environ Microbiol 70: 5037–5040. Shimoi H, Kitagaki H, Ohmori H, Imura Y & Ito K (1998) Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme This article is protected by copyright. All rights reserved.

Accepted Article

resistance. J Bacteriol 180:3381–3387. Srikrishnan S1, Chen W & Da Silva NA (2013) Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast. Biotechnol Bioeng 110: 275-285. Sun J, Wen F, Si T, Xu JH & Zhao H (2012) Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome. Appl Environ Microbiol 78: 3837-3845. Tanaka T, Yamada R, Ogino C & Kondo A (2012) Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol 95: 577-591. Tsai SL, DaSilva NA & Chen W (2013) Functional display of complex cellulosomes on the yeast surface via adaptive assembly. ACS Synth Biol 18:14-21. Tyo KE, Alper HS & Stephanopoulos GN (2007) Expanding the metabolic engineering toolbox: more options to engineer cells. Trends Biotechnol 25: 132-137. Tsai SL, Goyal G & Chen W (2010) Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production. Appl Environ Microbiol 76: 7514–7520. Van der Vaart JM, te Biesebeke R, Chapman JW, Toschka HY, Klis FM & Verrips CT (1997) Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins. Appl Environ Microbiol 63:615–620. Van Zyl JH, Den Haan R & Van Zyl WH (2014) Over-expression of native Saccharomyces cerevisiae exocytic SNARE genes increased heterologous cellulase secretion. Appl Microbiol Biotechnol 98:5567-5578. Washida M, Takahashi S, Ueda M & Tanaka A (2001) Spacer-mediated display of active lipase on the yeast cell surface. Appl Microbiol Biotechnol 56: 681–686. Wen F, Sun J & Zhao H (2010) Yeast surface display of trifunctional minicellulosomes for simultaneous saccharification and fermentation of cellulose to ethanol. Appl Environ Microbiol 76: 1251–1260. This article is protected by copyright. All rights reserved.

Accepted Article

Wentz AE & Shusta EV (2007) A novel high-throughput screen reveals yeast genes that increase secretion of heterologous proteins. Appl Environ Microbiol 73: 1189-1198. Wilde C, Gold ND, Bawa N, Tambor JH, Mougharbel L, Storms R & Martin VJ (2012) Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain. Appl Microbiol Biotechnol 95: 647-659. Yamada R, Yamakawa S, Tanaka T, Ogino C, Fukuda H & Kondo A (2011) Direct and efficient ethanol production from high- yielding rice using a Saccharomyces cerevisiae strain that express amylases. Enzyme Microb Technol 48: 393–396. Yamakawa S, Yamada R, Tanaka T, Ogino C & Kondo A (2010) Repeated batch fermentation from raw starch using a maltose transporter and amylase expressing diploid yeast strain. Appl Microbiol Biotechnol 87: 109–115. Yang J, Ang H & Lu JR (2012) Improving genetic immobilization of a cellulose on yeast cell surface for bioethanol production using cellulose. J Basic Microbiol 52: 1–9. Yi L, Gebhard MC, Li Q, Taft JM, Georgiou G & Iverson BL (2013) Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries. Proc Natl Acad Sci U S A. 110: 7229-7234. Zhang K, Nelson KM, Bhuripanyo K, Grimes KD, Zhao B, Aldrich CC & Yin J (2013) Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display. Chem Biol 20: 92-101.

Zlotnik H, Fernandez MP, Bowers B & Cabib E (1984) Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacteriol 159:1018–1026. Figure legends Figure 1. Creation of yeast cell factories using each approaches, surface display, synthetic biology and metabolic engineering. Figure 2. Schematic illustrations of cell surface display systems; N-terminal fusion and C-terminal fusion. This article is protected by copyright. All rights reserved.

Accepted Article

Figure 3. Schematic illustrations of mini-cellulosomes with different configurations reconstructed on a yeast cell surface. Graphical Abstract We review cell surface display for yeast cell factory from a synthetic biology perspective.

This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.