Microbial cell surface display technology provides a powerful platform for engineering proteins/peptides with enhanced properties. Compared to traditional intracellular and extracellular expression (secretion) systems, this technology avoids enzyme purification, substrate transfer processes and is an effective solution for enzyme instability. In the cell surface display platform, different enzymes can be located on the same cell surface and proximity can enhance their synergistic effects. Direct cell surface display is a simple and effective strategy. When multiple enzymes with synergistic effects are co-displayed, the ratio of various enzymes cannot be well controlled. Scaffold-mediated surface display systems can compensate for the shortcomings of direct display systems by shortening the transfer distance of substrates and enhancing the proximity effect between enzymes. However, the design is more complicated and cannot guarantee the assembly stability of the cell surface.

The efficiency of enzyme display on the cell surface is related to the attached anchoring proteins. Attempts have been made to improve cell surface display efficiency by screening for contrasting new anchoring proteins. Thirty-seven GPI-type anchor proteins were selected from different sources by computational prediction, fused to yEGFP, pre-screened by comparing fluorescence intensities, and further fused several potent fluorescent anchor proteins to β-glucosidase to determine enzyme activity. Finally, 6Kl from lactic acid Kluwer yeast was found to have higher transcript levels and superior target protein display efficiency. By comparing the display efficiency and enzyme activity of several display systems, the researchers adapted the a-lectin anchoring system using α-galactosidase as the target enzyme and fusing it directly to Aga1p to develop a new surface display system. The enzyme was also fused to conventional alpha-lectin and six other selected anchoring proteins. Comparison of enzyme activities showed that Aga1p , Dan4p and Sed1p have high display efficiency and are promising anchoring systems for immobilizing recombinant proteins to yeast surfaces. The display efficiency of the structural domains of the anchored proteins varied with the molecular weight of the displayed proteins. Enzyme expression can be further improved by optimizing the combination of promoter and signal peptide. The addition of a junction peptide of appropriate length between the anchor and target proteins appears to help separate the active portion of the protein from the cell wall, creating space for substrate entry and improving the activity of the displayed enzyme.

Hydrolysis of cellulose to glucose requires at least three cellulases: endoglucanase (EG), cellobiose hydrolase (CBH), and β-glucosidase (BGL). These three enzymes have been successfully shown to hydrolyze cellulose to glucose on the cell surface of yeast. the placement of EGII and TaCBHI in the same space (either on the cell surface or in the medium) facilitates fermentation of amorphous cellulose-based ethanol. Engineered yeasts demonstrated that these two enzymes, as well as two other enzymes acting on amorphous cellulose and crystalline cellulose, exhibited higher ethanol yields. Many attempts have been made to improve the fermentation process and introduce new lignocellulose degrading enzymes to further enhance cellulose degradation and product yield. Xylan, which is more readily degraded into monomers than cellulose through a pretreatment process, can be engineered yeast to achieve a one-step process of xylan assimilation with the gradual maturation of cell surface display engineering and heterologous expression of xylose metabolizing enzymes. Xylose-utilizing yeast can be constructed by introducing genes encoding enzymes related to the xylose assimilation pathway. A series of multifunctional micro hemicellulose vesicles were constructed for hydrolysis of arabinoxylan. Starch is a polymer of α-D-glucose and is abundantly present in many agricultural and industrial wastes. Degradation of starch feedstocks using surface-display engineered yeasts can not only produce biofuels but also improve process quality in the food industry. Lipases catalyze a variety of reactions and are widely used in industry. By using engineered yeast cells and displaying lipase on the cell surface as a recyclable biocatalyst, production costs can be reduced.

Inefficient production of heterologous proteins leads to lack of activity of engineered yeast strains, which may decrease even more with increasing recovery time. Most of the current studies are mainly at the laboratory level, and further research is needed to investigate whether the constructed engineered strains are suitable for industrialization on a large scale. To solve the above bottlenecks, in the future, we should develop more novel anchoring proteins to extend cell surface displayable sites and design new gene editing systems incorporating cell surface display to increase the copy number of target genes and simplify the plasmid transformation process. More biomaterials for encapsulating engineered yeast should be developed to increase the number of reuse, maintain the activity of display proteins, and further expand application areas. Construction of yeast cell surface display system by rational and systematic design. The combination of cell surface display technology, metabolic engineering and synthetic biology engineering can help to reduce the production of by-products and build a multifunctional “super yeast”.

One of the challenging tasks in the production of recombinant enzymes is the selection of yeast hosts. Although Saccharomyces cerevisiae has been the subject of extensive research and is currently one of the preferred hosts for the production of biologics, the yields typically obtained preclude its use for bulk enzyme production. Predicting the most suitable host remains impossible; instead, screening as many hosts as possible is the standard approach. As a rule of thumb, the host should be as closely taxonomically related to the donor organism as possible (i.e., the source of the enzyme gene). In this way, recombinant hosts should have somewhat similar requirements for cellular mechanisms (secretion, protein folding, etc.). Although it is desirable to be able to produce a wide range of enzymes, reality still suggests that a single host can only produce a limited share of the “protein universe”. Another consideration when selecting a host species is the level of endogenous protease, as it affects the yield and stability of the product. Obviously, a host strain that releases large amounts of protease during fermentation will result in less than optimal enzyme yields. The selected strains should be adapted from small-scale experiments to large-scale industrial production. This will make it possible to test thousands of strains to select the most suitable candidate for fermentation scale-up.