Recent progress in microbial cell surface display systems and their application on biosensors

Microbial cell surface display technology is a recombinant technology to express target proteins on the cell membrane,which can be used to redesign the cell surface with functional proteins and peptides.Bacterial and yeast surface display systems are the most common cell surface display systems of prokaryotic and eukaryotic proteins,that are widely applied as the core elements in the field of biosensors due to their advantages,including enhanced stability,high yield,good safety,expression of larger and more complex proteins.To further promote the performance of biosensors,the biomineralized microbial surface display technology was proposed.This review summarized the different microbial surface display systems and the biomineralized surface display systems,where the mechanisms of surface display and biomineralization were introduced.Then we described the recent progress of their applications on biosensors for different types of detection targets.Finally,the outlooks and tendencies were discussed and forecasted with the expectation to provide some general functions and enlightenments to this aspect of research.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Development of surface displaying system for heterologous protein expression in Candida tropicalis

The purpose of this study was to assess the potential application of cell surface display in Candida tropicalis.Surface display gene cassettes were constructed using five anchoring proteins from Saccharomyces cerevisiae,three of which[(suppression of exponential defect protein,SED1),(cell wall protein 2,CWP2)and(delayed anaerobic protein 4,DAN4)]were reported to show higher activity of heterologous proteins thanα-agglutinin(AGα1).The performance of yeast-enhanced green fluorescent protein(yeGFP)was evaluated using laser scanning confocal microscopy and flow cytometry.The results showed that the three anchoring regions(SED1,CWP2 and AGα1)successfully displayed yeGFP on the cell wall.To investigate the effect of the three anchoring proteins on the surface display of Rhizopus oryzaeα-amylase(ROA1)and Aspergillus aculeatusβ-glucosidase(BGL1)in C.tropicalis,we constructed surface display gene cassettes for ROA1 and BGL1,respectively.The strains containing the anchoring proteins SED1 and CWP2 showed higher activity of ROA1 and BGL1 than the strains containing the anchoring protein AGα1.The highest ROA1 and BGL1 activities of strains with SED1 were 6.37 U/g CDW and 7.93 U/g CDW,respectively,which were sixfold and eightfold higher than those of strain with AGα1.In addition,we also optimized signal peptides.The results indicated that signal peptides have an impact on enzyme activity.

Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic pocket and the application of peroxidase-coated yeast cell walls

The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods.Lignin peroxidase(LiP)from Phanerochaete chrysosporium is a hemecontaining lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules,including industrial dyes.This enzyme is therefore ideal as a starting point for protein engineering.Accordingly,we subjected two positions(165 and 264)in the environment of the catalytic Trp171 residue to saturation mutagenesis,and the resulting library of 104 independent clones was expressed on the surface of yeast cells.This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently.We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes(Evans blue,amido black 10B and Guinea green)and up to 13-fold higher catalytic activity.Additionally,cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis,achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst.LiPcoated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h.The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.

Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens(RIPPA)

CD4^(+)T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes.To study these responses,it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition.However,generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming.Here,we express human MHC alleles(HLA-DR4 and HLA-DQ6)as native,noncovalentαβdimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens.We demonstrate rapid,accurate identification of DQ6 ligands from pre-pro-hypocretin,a narcolepsy-related immunogenic target.We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes,and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4^(+)T cells from unexposed individuals carrying DR4 subtypes.Our method is optimized for immediate application upon the emergence of novel pathogens.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.