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Background

Overview

This project focuses on improving the yeast surface display system. Cell-surface display systems have been successfully developed in various microorganisms (Shibasaki, 2009), such as E. coli and Saccharomyces cerevisiae. The baker' s yeast is one of the most suitable hosts for the technology because of its rigid cell walls (around 110-200 nm wild) and its useful platform for protein production, which facilates the folding and glycosylation of expressed heterologous eukaryotic proteins. The rigid yeast cell wall is mainly composed of cross-linked β-1,3/ 1,6-glucans, mannoproteins, and chitin. Among these components, the β-1,6-glucan, though takes up little quantities of the system, plays an essential role in anchoring cell-wall proteins (Liu, 2016). Another important part in the display system is glucanase-extractable mannoproteins which contains glycosylphosphatidylinositol (GPI) anchors, such as agglutinin (Aga1 and Aga2). These GPI-anchored proteins can be utilized in anchoring foreign proteins by genetic engineering. The specific function of GPI-anchored proteins is described in later sections.

Figure 1 Architecture of the yeast cell wall. SMP, glucanase-extractablesurface-layer mannoprotein;
PP, SDS-extractable periplasmic protein (Seiji SHIBASAKI, et al. Analytical Science, 2009)

In recent years, yeast surface display system has been a hot spot in biotechnology. Applications of this technique exist in numerous fields. By anchoring enzymes, peptides or functional proteins on the cell wall, yeast surface display system can show the following advantages:

1. Heterologous eukaryotic protein expression can be accomplished simply by yeast cell propagation (Shibasaki, 2009), with few misfolding and incomplete modification;
2. Protein can be transported and immobilized by the secretory pathway, exocytosis (Liu, 2016). Thus, the expenditure and facilities needed for protein generation and enrichment in practical research or production can be reduced;
3. The yeast cell wall offers a solid surface to stabilize foreign proteins, which improves enzyme stability (Pack, 2002) in long-term storage and enable recycling in industrial processes;
4. Tethering of multiple synergetic enzymes on a single cell significantly shortens the enzyme-to-enzyme distance, preventing long-distance mass transfer of substrates, especially in high-solid fermentation;
5. Yeast cells can be measured through a flow cytometer, thus it makes high-throughput screenings more feasible and convenient (Shibasaki, 2009).

Regarding the significant advantages of yeast surface display system, it has a wide range of applications in research and industrial fields. For instance, in medicine, yeast surface display is employed to conduct high-throughput antibody screening and manufacturing oral vaccine for precaution (Chao, 2006), diagnostics and therapeutics. Additionally, since yeasts play a critical role in the commercial production of fuel and fermentation (Liu, 2016), this technique is utilized to display amylolytic enzymes, lipase, and cellulolytic enzymes to act as a whole-cell biocatalyst. Moreover, it can also be designed as a heavy metal-absorbent or a non-invasive sensor and monitor.

Our Goal

Despite several extraordinary advantages presented above, we still envision some prospects for this technique. Up to now, only separate components, such as monomer protein subunits, has been displayed. No assembly has been done. If the polymer could be displayed as a whole on yeast cell wall, a lot more could be done. For example, the detection of some small molecules relies on the integrity of the polymer. Displaying polymers on yeast would allow high-throughput screening to be realized. The display of complex enzymatic pathways could also be accomplished using this new system. Currently, the types of co-displayed enzymes are limited. Our solution is to create an extracellular scaffold for enzymes to interact to each other. This would greatly increase the variety and quantity of displayed enzymes.