Dataset Information

Development of novel surface display platforms for anchoring heterologous proteins in Saccharomyces cerevisiae.

ABSTRACT:

Background

Cell surface display of recombinant proteins has become a powerful tool for biotechnology and biomedical applications. As a model eukaryotic microorganism, Saccharomyces cerevisiae is an ideal candidate for surface display of heterologous proteins. However, the frequently used commercial yeast surface display system, the a-agglutinin anchor system, often results in low display efficiency.

Results

We initially reconstructed the a-agglutinin system by replacing two anchor proteins with one anchor protein. By directly fusing the target protein to the N-terminus of Aga1p and inserting a flexible linker, the display efficiency almost doubled, and the activity of reporter protein ?-galactosidase increased by 39%. We also developed new surface display systems. Six glycosylphosphatidylinositol (GPI) anchored cell wall proteins were selected to construct the display systems. Among them, Dan4p and Sed1p showed higher display efficiency than the a-agglutinin anchor system. Linkers were also inserted to eliminate the effects of GPI fusion on the activity of the target protein. We further used the newly developed Aga1p, Dan4p systems and Sed1p system to display exoglucanase and a relatively large protein ?-glucosidase, and found that Aga1p and Dan4p were more suitable for immobilizing large proteins.

Conclusion

Our study developed novel efficient yeast surface display systems, that will be attractive tools for biotechnological and biomedical applications.

SUBMITTER: Yang X

PROVIDER: S-EPMC6525377 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Publications

Development of novel surface display platforms for anchoring heterologous proteins in Saccharomyces cerevisiae.

Yang Xiaoyu X Tang Hongting H Song Meihui M Shen Yu Y Hou Jin J Bao Xiaoming X

Microbial cell factories 20190518 1

<h4>Background</h4>Cell surface display of recombinant proteins has become a powerful tool for biotechnology and biomedical applications. As a model eukaryotic microorganism, Saccharomyces cerevisiae is an ideal candidate for surface display of heterologous proteins. However, the frequently used commercial yeast surface display system, the a-agglutinin anchor system, often results in low display efficiency.<h4>Results</h4>We initially reconstructed the a-agglutinin system by replacing two anchor ...[more]

PMID: 31103030

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Project description:BACKGROUND: Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-beta-N-acetylglucosaminidase (Mbg), as the anchor protein. RESULTS: Homology searching in the B. thuringiensis YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM1, or LysM2 were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM1 was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (Mbgn)2 anchor, without radically altering its catalytic activity. CONCLUSION: Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy presented here could be applicable in other bacteria for developing this type of system.

Project description:BackgroundFor industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.ResultsTwo Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the alpha-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40 degrees C at pH8.0, they retained over 90% activity after incubation at 60 degrees C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.ConclusionThe LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.

Dataset Information

Development of novel surface display platforms for anchoring heterologous proteins in Saccharomyces cerevisiae.

Background

Results

Conclusion

Publications

Development of novel surface display platforms for anchoring heterologous proteins in Saccharomyces cerevisiae.

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