Existing Part: Appa phytase( BBa_K912001, iGEM12_Northwestern)

New Improved Part: pYES2-Hyg-GAL1p-AppA-SED1-RPL41Bt(BBa_K4870016)

Summary:

In this project, to expand the application of appa phytase( BBa_K912001) in feed, we inserted the AppA gene into the plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt (BBa_K4870015) and transformed into yeast cells.In addition, we utilized yeast cell surface display technology and supplemented the characterization of AppA phytase activity displayed on yeast under different pH and temperature conditions. For reaction temperature, the activity of this phytase increased with rising temperature, with the highest activities detected at 55 °C.

Usage and Biology:

We transformed the plasmid into yeast and fused the phytase encoding gene with the anchoring protein encoding gene on the cell surface to produce yeast single cell protein with phytase activity, which can be added to feed to enhance animal nutrition and absorption^[1],[2].

In this project, to expand the application of appa phytase( BBa_K912001) in feed, we utilized yeast cell surface display technology and supplemented the characterization of AppA phytase activity displayed on yeast under different pH and temperature conditions. For reaction temperature, the activity of this phytase increased with rising temperature, with the highest activities detected at 55 °C

Figure 1. Design diagram of this project. Image by LIU JINLE.

Cultivation, Purification and SDS-PAGE

The positive transformant was inoculated and plasmid was extracted using a commercial miniprep kit. 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 plasmid into BY4741 competent cells, followed by incubation at 30 °C for 48 h. The colony PCR identification confirmed that the plasmid was successfully transfected into BY4741 cells. Sequencing of the transformants verified the correct sequence of the AppA target fragment.

Figure 2. Results of AppA display plasmid transformation of yeast cells.

We selected a positive transformant and cultured it 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. The SDS-PAGE result showed that the AppA was successfully displayed on the yeast cell surface (Figure 3).

Figure 3. AppA SDS-PAGE and phytase activity under different (A) pH and (B)

temperature conditions.

Characterization/Measurement

Where the SED1 protein targets AppA to the yeast cell wall. After transforming the recombinant plasmid into Saccharomyces cerevisiae cells and inducing expression galactose, we obtained AppA phytase displayed on the yeast surface. Enzyme activity was determined under different pH and temperature conditions. We found that AppA phytase activity initially increased with rising pH, reaching a maximum at pH 5, then decreasing as pH continued to rise. For reaction temperature, AppA phytase activity increased with temperature, with the highest activity detected at 55°C(Figure 3)

References:

[1] Chen X, Xiao Y, Shen W, Govender A, Zhang L, Fan Y, Wang Z. Display of phytase on the cell surface of Saccharomyces cerevisiae to degrade phytate phosphorus and improve bioethanol production. Appl Microbiol Biotechnol. 2016 Mar;100(5):2449-58. doi: 10.1007/s00253-015-7170-4.

[2] Navone L, Vogl T, Luangthongkam P, Blinco JA, Luna-Flores C, Chen X, von Hellens J, Speight R. Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris. Microb Cell Fact. 2021 Jan 7;20(1):8.