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Background

Overview

This year our project focuses on improving the yeast surface display system.Cell-surface display systems have been successfully developed in various microorganisms, such as Escherichia coli. While, yeast is one of the most suitable host strains for this arming technology, because of its rigid cell walls (around 110–200 nm wild) and useful platform for protein production which allows the folding and glycosylation of expressed heterologous eukaryotic proteins. The rigid yeast cell wall was mainly constituted by cross-linked β-1, 3/1, 6-glucans, mannoproteins, and chitin. Among these components, the β-1,6-glucan，though takes up a little quantities of the system, plays an essential role in anchoring cell-wall proteins. 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 section.

Figure 1 Architecture of the yeast cell wall. SMP, glucanase-extractable
surface-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 of research. Application of this technique exists in numerous fields. With 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, with few misfolding and incomplete modification;
(2) Protein can be transported and immobilized by the secretory pathway, exocytosis. 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 stable foreign proteins that can improve enzyme stability in long-term storage and 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 cell can be measured through a flow cytometer, thus it makes high-throughput screenings more feasible and convenient.
Regarding the significant advantages of yeast surface display system, it has wide range of applications in nowadays research and industrial fields. For instance, in medical related field, yeast surface display is employed to conduct high-throughput antibody screening and manufacturing oral vaccine for precaution, diagnostics and therapeutics. Additionally, since yeast play a critical role in the commercial production of fuel and fermentation, this technique is utilized to display amylolytic enzymes, lipase, cellulolytic enzymes as a whole-cell biocatalyst. Moreover, it can also be designed as a heavy metal-absorbent or a non-invasive sensor and monitor.

Our Prospects

Despite several extraordinary advantages have be presented above, we still envision some prospect for this technique.Up to now, only separate component, such as a monomer protein subunit, has been displayed. No assembly has been done. If the polymer could be displayed as a whole on yeast cell wall, a lot 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. And display complex enzymatic pathways. Currently, the types of co-displayed enzymes are limited. Our solution is to create an extracellular scaffold for enzymes to attach on. This would greatly increase the types and quantity of displayed enzymes.

[1]Liu, Z., et al. "Recent advances in yeast cell-surface display technologies for waste biorefineries." Bioresource Technology 215(2016):324.
[2]Shibasaki, S, H. Maeda, and M. Ueda. "Molecular display technology using yeast--arming technology. " Analytical Sciences the International Journal of the Japan Society for Analytical Chemistry 25.1(2009):41.
[3]Chao, G., et al. "Isolating and engineering human antibodies using yeast surface display. " Nature Protocols 1.2(2006):755.

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