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Shibasaki, S.;  Ueda, M. Molecular Display Technology. Encyclopedia. Available online: https://encyclopedia.pub/entry/40146 (accessed on 01 January 2025).
Shibasaki S,  Ueda M. Molecular Display Technology. Encyclopedia. Available at: https://encyclopedia.pub/entry/40146. Accessed January 01, 2025.
Shibasaki, Seiji, Mitsuyoshi Ueda. "Molecular Display Technology" Encyclopedia, https://encyclopedia.pub/entry/40146 (accessed January 01, 2025).
Shibasaki, S., & Ueda, M. (2023, January 13). Molecular Display Technology. In Encyclopedia. https://encyclopedia.pub/entry/40146
Shibasaki, Seiji and Mitsuyoshi Ueda. "Molecular Display Technology." Encyclopedia. Web. 13 January, 2023.
Molecular Display Technology
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This entry is adapted from the peer-reviewed paper 10.3390/microorganisms11010125

Molecular display technology or cell-surface engineering is a biotechnological method of genetic engineering that is focused on the cell surface.

yeast genetic engineering molecular display technology

1. Introduction

In biochemical studies, yeasts are considered to be a representative model of eukaryotic microbes. The budding yeast Saccharomyces cerevisiae contains only 6611 genes (https://www.yeastgenome.org, accessed on 16 December 2022); however, it has been used for investigating gene and protein functions in recent molecular biology studies [1][2][3][4][5].
Moreover, the use of S. cerevisiae as catalysts in various fermentation industries has a longer history than their application in modern bioscience and biotechnology. For instance, yeasts have been employed in the production processes of fermented foods, such as Japanese sake [6][7], beer [8], bread [9][10], and miso [11][12]. Thus, S. cerevisiae has been related to our diet for a long time and gained a generally regarded as safe (GRAS) status [13][14], although a few strains are pathogenic [15]. Since the advent of genetic engineering, S. cerevisiae has been used for producing valuable compounds. Several recombinant proteins have also been developed as pharmaceuticals using yeast cells [16][17]. Given their usefulness and safety, S. cerevisiae can help us achieve sustainable development.
In this century, biotechnology is expected more than ever to solve socioeconomic issues. Although industrialization has made human life convenient, it has also given rise to environmental issues, such as global climate change, food shortage, and pollution. To overcome these problems, the United Nations proposed 17 Sustainable Development Goals (SDGs) for maintaining human health and establishing a sustainable society [18]. Natural resources or products form the basis of and are consumed during the economic activities in developing and developed countries, causing industrial and environmental problems that need to be mitigated. Considering that biotechnology has improved the quality and scale of industrial and agricultural production, it may also provide several opportunities for sustainable development. The latest biotechnology development in manufacturing not only improves production efficiency, but also promotes international trade and cooperation toward mutual development [19]. Among various biotechnologies, microbial biotechnology is expected to achieve SDGs [20]. Microbiology and microbial technology are considered the key to achieving SDGs via the eradication of infectious diseases, provision of clean water, food security, maintenance of terrestrial and marine biodiversity, and utilization of biofuels [21].

2. General Description of Molecular Display Technology

Molecular display technology or cell-surface engineering is a biotechnological method of genetic engineering that is focused on the cell surface. The phage display system proposed by Smith has the longest history among molecular display systems [22]. It was employed in searching for a clone that can bind to a target compound or investigating protein interactions and is still widely used today [23][24]. A phagemid vector encoding a foreign protein to be displayed as a fusion of the coat protein can be introduced into a phage, resulting in a library consisting of 1012 clones. Next, the panning process is conducted to select positive clones that can bind to target molecules from this phage library; this process includes immobilization of the target molecule on a solid phase, incubation of a library and the target molecule, removal of bound phages from solid phages, infection of Escherichia coli with the recovered phage, and amplification of the clones. This sequential panning cycle should be performed several times. In addition, bacterial cells have been developed and were shown to improve the phage display method, which uses complicated panning processes to isolate a clone capable of binding to a target. For example, surface display systems of foreign proteins using Lactococcus lactis [25], Staphylococcus aureus [26], and E. coli [27] as the host cell have been developed. In addition, unlike phage display systems, bacterial display systems were applied not only in the selection of protein clones that can bind to a target compound, but also in the establishment of whole-cell biocatalysts coupled with metabolic reaction in host cells [28].
Yeasts have been employed in molecular display technology for 20 years [29][30][31]. The yeast S. cerevisiae is useful as a host microorganism in genetic manipulation because it can produce and glycosylate foreign proteins derived from other eukaryotes. This yeast species also has the advantage of high-density cultivation in various media at a low cost. In addition, this yeast not only displays proteins derived from other eukaryotes but also different proteins on its cell surface, i.e., “co-display” [32]. As another characteristic of host cells, several auxotrophic markers can be used in the genetic manipulation of yeast cells for producing different recombinant proteins. Furthermore, flow cytometry or high-throughput screening is applicable for selecting target protein-displaying yeast cells [33][34][35]. Thus, molecular display or cell-surface engineering using the yeast cell surface has many important benefits and practical applications. Yeasts capable of displaying foreign proteins, especially S. cerevisiae, are called “arming yeasts” [36][37][38][39][40]. The principle of molecular display using yeasts and its applications to achieve sustainable development are describe in the subsequent sections.

3. Principle of Molecular Display Technology Using Yeasts

Regardless of the type of host cells selected for molecular display, the anchoring protein must be valid and effective. For example, OmpA has been investigated and used as an anchoring protein to display a foreign protein on the cell surface of E. coli [41][42]. It is usually used as a lipoprotein, which is fused to residues 46–159 of the OmpA porin protein family to anchor to the E. coli cell wall envelope. Moreover, the cA domain of AcmA (a major autolysin from L. lactis) [25] and PgsA from Bacillus subtilis [43] have shown efficacy in displaying several foreign proteins on the cell surface of Lactobacillus sp.
In a S. cerevisiae molecular display system, several anchoring proteins can be used to immobilize the target protein (Figure 1). The Flo1 protein can anchor the target protein in two ways. It is produced and attached on the yeast cell surface for flocculation. In molecular display technology, the target protein to be displayed on the yeast cell surface is usually fused at its N-terminal to the C-terminal of the Flo1 protein. Moreover, Aga1-Aga2 proteins, which are also used for displaying the target protein, are favorable because they can display a C-terminal-free protein on the yeast cell surface. If the active site of the target protein is located in the N-terminal, or if its C-terminal conformation does not affect the function of the target protein, α-agglutinin can be used.
Saccharomyces cerevisiae has a thick, hard cell wall that consists of β-linked glucans and mannoproteins. The cell wall is located outside the plasma membrane and consists of an internal skeletal layer of glucan composed of β-1,3- and β-1,6-linked glucose and a fibrillar or brush-like outer layer composed predominantly of mannoproteins [44]. There are two types of mannoproteins in the thick cell wall [45]. One is loosely bound to the cell wall with non-covalent bonds and can be extracted using sodium dodecylsulfate (SDS). The other mannoprotein can be extracted by using β-1,3- or β-1,6-glucanase. Cell wall proteins illustrated in Figure 1 are glucanase-extractable mannoproteins covalently linked with the glucan layer of the cell wall.
Since the development of yeast molecular display systems, α-agglutinin has been widely investigated and used as an anchoring protein [46][47][48]. Mating-type alpha cells have α-agglutinin protein on their cell surface, which functions during mating with other types of S. cerevisiae cells. This protein is covalently bound to the cell wall, causing the target protein to be fused and stably displayed on the cell surface. Although α-agglutinin itself has a predicted length of 650 amino acids before processing, genetic engineering allows the fusion of the target protein to the C-terminal half (320 amino acids) of α-agglutinin as a cell wall anchor. In the genetic preparation for molecular display, a gene encoding foreign protein is placed between a gene encoding secretion signal sequence and a gene encoding the C-terminal half of α-agglutinin (Figure 2). This genetic construct is expressed under a suitable promoter sequence in a plasmid when it is successfully incorporated by the host yeast cells. In the following sections, several applications of molecular display systems of S. cerevisiae with α-agglutinin are described.
Microorganisms 11 00125 g001
Figure 1. Molecular display systems in Saccharomyces cerevisiae. A target protein is immobilized by several types of anchoring proteins. (a) a-agglutinin-based display system. (b) Flo1p-based display system. (c) α-Agglutinin-based display system. Figures were adapted from [47].
Microorganisms 11 00125 g002
Figure 2. Fusion gene construction for cell-surface display of metal-binding protein and peptide on yeast cell surface. Secretion signal sequence is necessary for extracellular transport of a fusion protein.

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