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.

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.

Surface display of carbonic anhydrase on Escherichia coli for CO_(2) capture and mineralization

Mineralization catalyzed by carbonic anhydrase(CA)is one of the most promising technologies for capturing CO_(2).In this work,Escherichia coli BL21(DE3)was used as the host,and the N-terminus of ice nucleation protein(INPN)was used as the carrier protein.Different fusion patterns and vectors were used to construct CA surface display systems forα-carbonic anhydrase(HPCA)from Helicobacter pylori 26695 andα-carbonic anhydrase(SazCA)from Sulfurihydrogenibium azorense.The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity,while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system.The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA.Cell frac-tionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell’s outer membrane as a fusion protein.The enzyme activity of the surface display strain E-22b-I RL S(11.43 U⋅mL^(−1) OD 600−1)was significantly higher than that of the intracellular expression strain E-22b-S(8.355 U⋅mL^(−1) OD 600−1)under optimized induction conditions.Compared with free SazCA,E-22b-I RL S had higher thermal and pH stability.The long-term stability of SazCA was also significantly improved by surface display.When the engineered strain and free enzyme were used for CO_(2) mineralization,the amount of CaCO_(3) deposition catalyzed by the strain E-22b-I RL S on the surface(241 mg)was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S(173 mg).These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO_(2) capture and mineralization.

Isolation of Goose-origin scFv Antibodies Against Goose Parvovirus from Bacterial Display Antibody Libraries

Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.

Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria

Phenylketonuria(PKU),a disease resulting in the disability to degrade phenylalanine(Phe)is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity.As an potential alternative to a protein-restricted diet,oral intake of engineered probiotics degrading Phe inside the body is a promising treatment,currently at clinical stage II(Isabella,et al.,2018).However,limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s activity.Here,we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase(PAL)as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917(EcN)which overcomes the transportation problem.The metabolic engineering strategy was also combined with strengthening of Phe transportation,transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia.Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah^(F263S)PKU mouse model reduced blood Phe concentration by 44.4%compared to the control Ec N,independent of dietary protein intake.TYS8500 shows great potential in future applications for PKU therapy.

Cell surface protein engineering for high-performance whole-cell catalysts

Cell surface protein engineering facilitated by accumulation of information on genome and protein structure involves heterologous production and modifica- tion of cell surface proteins using genetic engineering, and is important for the development of high-performance whole-cell catalysts. In this field, cell surface display is a major technology by exposing target proteins, such as enzymes, on the cell surface using a cartier protein. The target proteins are fused to the carrier proteins that transport and tether them to the cell surface, as well as to a secretion signal. This paper reviews cell surface display systems for prokaryotic and eukaryotic cells from the perspective of carrier proteins, which determine the number of displayed molecules, and the localization, size, and direction （N- or C-terminal anchoring） of the passengers. We also discuss advanced methods for displaying multiple enzymes and a new method for the immobilization of whole-cell catalysts using adhesive surface proteins.

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.