Overview
Our experimental results mainly include:
(1) Construction of plasmids for surface display of phytase,
(2) Obtaining surface-displayed phytase in Saccharomyces cerevisiae,
(3) Determining phytase activity under different pH and temperature conditions.
1. Construction of plasmids for surface display of phytase
We first constructed the following two plasmids:
Plasmid 1: pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt,
Plasmid 2: pYES2-Hyg-GAL1p-α-An_phy33-SED1-RPL41Bt.
To construct plasmid 1, we used the E. coli DH5α genomic DNA as a template to amplify the AppA sequence by polymerase chain reaction (PCR) (Figure 1A). Then, the AppA sequence was inserted into the SphⅠ and AvrⅡ sites of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by restriction endonuclease digestion and linkage.
To construct plasmid 2, we used a synthetic plasmid containing the An_phy33 sequence as a template to amplify the An_phy33 sequence by PCR (Figure 1A). Then, the An_phy33 sequence was inserted into the SphⅠ and AvrⅡ sites of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by restriction endonuclease digestion and linkage.
(Note: The plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt contains GAL1p yeast promoter, MF-alpha-1 signal peptide, yeast surface anchoring protein gene SED1, and RPL41Bt yeast terminator.)
Subsequently, the plasmids 1 and 2 were transferred into the E. coli DH5α competent cells, respectively. As shown in Figure 1B, transformants were successfully grown after overnight culture. The mapping of the recombinant plasmids is illustrated in Figure 1C-D. After colony PCR verification, the recombinants had the expected bands (1293 bp for plasmid 1, and 1380 bp for plasmid 2), indicating successful transformation of both plasmids (Figure 1E).
Figure 1 Construction and transformation of the plasmids.
2. Obtaining surface-displayed phytase in Saccharomyces cerevisiae BY4741
Positive transformants were inoculated and plasmids were extracted using a commercial miniprep kit for subsequent transformation of Saccharomyces cerevisiae competent cells. Before the transformation, the BY4741 strain was inoculated and prepared in a competent state. Then, the lithium acetate (LiAc) transformation method was used to transform linearized plasmids 1 and 2 into BY4741 competent cells, respectively, followed by incubation at 30°C for 48 h.
As shown in Figure 2A, transformants were successfully cultured on YPD plates. After colony PCR identification, the two transformants had the expected bands (1293 bp for plasmid 1, and 1380 bp for plasmid 2), indicating that the plasmids were successfully transfected into BY4741 cells (Figure 2B). These transformants were then sequenced, confirming the AppA and An_phy33 target fragment sequences were correct (Figure 2C-D).
Figure 2 Results of plasmid transformation of yeast cells.
We selected positive transformants and cultured them to the logarithmic growth period. Galactose was then added to induce the promoter, and yeast cells displaying phytase on the surface were obtained. Cells were pelleted by centrifugation and washed by resuspension in 50 mM sodium acetate buffer (pH 5.0) A portion of the suspension was sonicated and then centrifuged to obtain lysate supernatant and precipitate containing cell wall-bound phytase. As shown in Figure 3, both AppA and An_phy33 were successfully displayed on the yeast cell surface for subsequent activity assays.
Figure 3 SDS-PAGE results of APPA and An_phy33 expression.
3. Determining phytase activity under different pH and temperature conditions.
We performed enzyme activity assays on yeast surface-displayed phytase and phytase lysate precipitate, respectively. We found that the lysed precipitated phytase exhibited higher activity, so this form was used to determine phytase activity under different pH and temperature conditions. As shown in Figure 4, in the pH range of 1-8, the activities of the two phytases initially increased with rising pH, reaching maxima at pH 5-6, then declining as pH continued to rise. AppA phytase showed peak activity at pH 5, while An_phy33 phytase had maximal activity at pH 6. For reaction temperature, the activities of both phytases increased with rising temperature, with the highest activities for both enzymes detected at 55 °C.
Figure 4 Phytase activity under different pH and temperature conditions.
In summary, we successfully constructed two plasmids that can display phytase on the surface of yeast cells, obtained surface-displayed phytase in Saccharomyces cerevisiae BY4741, and tested the activity of phytase under different pH and temperature conditions. Our experimental results confirmed fusion expression of the phytase gene with a yeast cell wall anchor protein gene enables the successful display of active phytase on the yeast cell surface.