Functional improvement of natural Saccharomyces cerevisiae yeast strains by cell surface molecular engineering

Abstract

Background: Cellular boundaries of microorganisms can be modified by the expression in the cell wall of specific proteins endowed with relevant properties, improving their functional performance. So far, the surface display (SD) technique had been widely employed in the yeast Saccharomyces cerevisiae, but it was limited to few laboratory strains and never explored in sauvage strains, i.e., isolated from natural environment, which are featured by higher levels of genetic variability, leading to peculiar phenotypic traits of possible advantage in biotechnology.

Results: In this work, a series of plasmids performing SD in natural yeast strains have been generated and further characterized by multiple functional and biochemical assays, providing the first experimental evidence that natural strains of S.cerevisiae can be genetically modified to express on their cell wall a protein-of-interest, which retains its biological competence. Interestingly, data further demonstrated that engineered strains expressing (transiently or stably) metal-binding proteins or peptides on cell surface exhibit significantly enhanced metal adsorption properties.

Conclusions: The molecular tools presented here can be very useful for yeast research community, as the plasmids efficiently support the surface engineering in virtually all S.cerevisiae strains, independently from either genetic background, source, or applications (wine, beer, bread). Overall, data strongly suggest that, upon genetic modification, S. cerevisiae strains isolated from natural environments could serve as promising platforms for biotechnological applications, as heavy metals removal or enzymes immobilization. Importantly, the strains investigated here represent only a small fraction of the multitude of S. cerevisiae strains present in nature yet to be isolated.

Keywords: Saccharomyces cerevisiae; Bioremediation; Protein engineering; Yeast surface display.

Fig. 1

Yeast Surface Display (YSD) plasmids construction and maps. Yeast multicopy plasmid pYES2 was sequentially modified by the insertion of the MFα1 sequence fused to the FLAG tag (1), followed by the cloning of the cell wall anchor sequence (SAG1-C_ter) (2). In the resulting vector, between MFα1-FLAG and SAG1-C_ter was (in-frame) cloned the coding sequence of either yeast metallothionein (yCup1), GFP protein, or exa-Histidine peptide (6xHis), generating pGAL-YSD plasmids series (3). The substitution of the pGAL1 (inducible) with the pGAP (constitutive) promoter originated the pGAP-YSD plasmids, constitutively expressing the chimeric proteins in yeast cells (4). Then, the nutritional yeast recessive URA3 marker was substituted by the dominant NrsR marker, to use pYSD plasmids in natural strains (5). Two additional copies of yCup1 protein were cloned in the pYSD-yCup1 plasmid to generate the chimeric protein with 3 repeats of yCup1 fused as tandem (6). Plasmid functional elements and their modifications are indicated by colors. 2µ ori: yeast replication; pUC ori: E.coli replication; AmpR: E.coli selection marker; CYCter: yeast transcription termination sequence. The name of each plasmid is reported, accordingly to Supplementary Table ST2. Image was created with BioRender.com

Fig. 2

Analysis of the YSD plasmids in yeast laboratory strain. (a) Total proteins from CENPK laboratory cells (two independent clones, #1–2), carrying either pGAP-YSD-GFP, pGAP-YSD-yCup1, pGAP-YSD-6xHis plasmids, or the empty vector as control (WT), were subjected to Western blot assay using the anti-FLAG antibody and with anti-GAPDH antibody. Images are representative of three independent experiments. (b) CENPK cells transformed as in (a), were analyzed by immunofluorescence microscopy to visualize the localization of the fusion proteins. For each strain, micrographs are reported of the differential interference contrast (DIC), the immunofluorescence signal (AF555), the nuclear DNA staining (DAPI), and the merged image. Images are representative of three biological replicates for each yeast strain. Scale bar: 5 μm. (c) Growth curves of yeast CENPK cells transformed with either pGAP-YSD-yCup1 (red) or empty (black) plasmids, incubated in SD medium (empty dots), or SD added with 2mM of CuSO₄ (filled dots). Growth rate was monitored for 72 h by OD₆₀₀ measurement (left panel). The OD₆₀₀ final values (72 h) are also reported as graph (right panel). Each column is the mean ± SEM of 4 independent experiments (n = 4, *p < 0,05, ONE WAY Anova with Kruskal-Wallis test was applied for statistical analysis). (d) Living CENPK cells carrying the pYSD-GFP plasmid were directly observed by confocal microscopy. Green fluorescence signal (GFP), differential interference contrast (DIC) micrograph, and merge are reported. Image is representative of three biological replicates; scale bar = 5 μm

Fig. 3

Analysis of the YSD plasmids in natural yeast strains. (a) Total proteins from 8 natural yeast strains (IB1-IB8), carrying the pYSD-yCup1 plasmid, were analyzed by Western blot as indicated in Fig. 2. (b) Western blot assay performed for the IB1-IB3 natural strains transformed with pYSD-6xHis. The GAPDH protein was used as loading control. Images are representative of three independent experiments. (c) Yeast IB1-IB3 strains carrying either pYSD-yCup1, or pYSD-6xHis plasmids, were analyzed by immunofluorescence microscopy to visualize the localization of fusion proteins. For each strain, micrographs are reported of the differential interference contrast (DIC), the immunofluorescence signal (AF555), the nuclear DNA staining (DAPI), and the merged image. Images are representative of three biological replicates for each yeast strain. Scale bar = 5 μm

Fig. 4

Metal binding by yeast strains expressing chimeric proteins on the outer surface. (a) 5 × 10⁸ yeast cells of each indicated strain (parental (WT) or carrying the pYSD-6xHis plasmid), were used for Ni²⁺ binding experiments as described, and ICP-EOS assays were performed to quantify the nmoles of metal ions, reported in the graphs. The ratio between values of modified and parental strains (YSD/WT) is indicated for each strain. (b) Similar experiments were performed using double (10⁹) amount of yeast cells. (c) As in (a), 5 × 10⁸ yeast cells, either parental (WT) or carrying the pYSD-yCup1 plasmid, were used for Cu²⁺ binding experiments, and Cu²⁺ ions quantification data are presented as nmoles of Cu²⁺ normalized to mg of CDW. In all panels, each column represents the mean ± SEM of 5 independent experiments (n = 5; **p < 0,01, Mann Whitney T-test was applied for statistical analysis)

Fig. 5

Analysis of genetically modified yeast cells. (a) Yeast IB1- and IB2-derivative strains, carrying one chromosomal copy of the YSD-yCup1 transgene replacing ADE2 gene, were analyzed by immunofluorescence microscopy to visualize the localization of the fusion protein. For each strain, micrographs are reported of the differential interference contrast (DIC), the immunofluorescence signal (AF555), the nuclear DNA staining (DAPI), and the merged image. Images are representative of three biological replicates for each yeast strain. Scale bar: 5 μm. (b) 5 × 10⁸ yeast cells of the IB1 and IB2 strains, either unmodified (WT), or carrying one chromosomal copy of the yCup1 chimeric transgene (ade2D::YSD-yCup1), were checked for Cu²⁺ binding by ICP-EOS and quantification data are reported in the graphs (as nmoles of Cu²⁺ / mg CDW). The ratio between values of modified and parental strains (YSD/WT) is indicated for each strain. Each column represents the mean ± SEM of 5 independent experiments (n = 5; *p < 0,05, **p < 0,01, Mann Whitney T-test was applied for statistical analysis)

Fig. 6

Analysis of IB1 yeast cells expressing multiple binding units (a) Yeast IB1 cells either transformed with the pYSD-3x-yCup1 plasmid, or carrying one chromosomal copy of the YSD-3x-yCup1 transgene in the ADE2 locus, were analyzed by immunofluorescence microscopy to visualize the localization of the fusion protein. For each strain, micrographs are reported of the differential interference contrast (DIC), the immunofluorescence signal (AF555), the nuclear DNA staining (DAPI), and the merged image. Images are representative of three biological replicates for each yeast strain. Scale bar: 5 μm; (b) 5 × 10⁸ yeast cells of either unmodified IB1 strain (WT), cells expressing triple-tandem yCup1 chimeric protein by multicopy plasmid (pYSD-3x-Cup1), or by single chromosomal copy of the transgene (ade2D::YSD-3x-yCup1), and Cu²⁺ binding was evaluated by ICP-OES, as previously described. Each column represents the mean ± SEM of 4 independent experiments (n = 4; *p < 0,05, Mann Whitney T-test was applied for statistical analysis)