Overview：
The growing global livestock population has led to shortages of traditional feed protein and raw materials, an urgent issue for the livestock and breeding industry. Plants also contain antinutrient phytic acid which inhibits nutrient absorption in animals. An alternative protein source is needed. This work aims to prepare yeast single-cell protein with phytase activity by fusing a phytase gene to a cell wall anchor protein. Adding this to feed could improve nutrition and absorption for livestock and poultry, benefiting feed producers.
Figure 1 Design diagram of this project. Image by LIU JINLE.
Cycle 1: Generation of surface-displayed AppA of yeast
Design 1:
We first obtained the phytase gene AppA (Gene ID:946206, NP_415500.1) from E. coli at the NCBI website (https://www.ncbi.nlm.nih.gov/). Then, using the pYES2 plasmid as a backbone, we designed an expression frame for yeast cell surface display phytase, i.e., to link the yeast promoter GAL, MF-alpha-1 signal peptide, AppA gene, yeast surface anchoring protein gene SED1, and RPL41Bt yeast (Figure 2). Finally, through restriction endonuclease digestion and linkage, we constructed the APPA display plasmid: pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt.
Figure 2 Expression frame of the AppA display plasmid
Build 1:
To construct the plasmid pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt, we used the E. coli DH5α genomic DNA as a template to amplify the AppA sequence by PCR. 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.
The plasmid was then transferred into the E. coli DH5α competent cells. The result showed that transformants were successfully grown after overnight culture. After colony PCR verification, the recombinants had the expected bands (1293 bp), indicating the successful transformation of this plasmid.
Figure 3 Construction of the AppA display plasmid
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 4 Results of AppA display plasmid transformation of yeast cells.
Test 1:
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 5).
Then, the activity assays were performed 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 5, the activity of the AppA phytase initially increased with rising pH, reaching maxima at pH 5, then declining as pH continued to rise. For reaction temperature, the activity of the AppA phytase increased with rising temperature, with the highest activity detected at 55 °C.
Figure 5 Expression and enzyme activity test results of AppA.
Learn 1:
The results of our experiments indicate that the AppA phytase displayed on the surface of yeast cells is active. The highest enzyme activity was observed at pH 5, or a reaction temperature of 55 °C. To further improve the activity of phytase, we predicted the ancestral enzyme gene sequence of AppA, An_phy33, which was used for the next round of plasmid construction, protein expression, and enzyme activity testing.
Cycle 2: Generation of surface-displayed An_phy33 of yeast
Design 2:
We obtained a series of ancestral enzyme sequences of E. coli phytase AppA at the website (https://loschmidt.chemi.muni.cz/fireprotasr/). Prediction using DeepSTABp (https://csb-deepstabp.bio.rptu.de/) revealed that the An_phy33 sequence had the highest melting temperature Tm of 87 °C compared to 51 °C for wild-type AppA, so we chose to use this sequence for this round of experiments.
Similar to the previous round of expression frames, we link the yeast promoter GAL, MF-alpha-1 signal peptide, An_phy33 gene, yeast surface anchoring protein gene SED1, and RPL41Bt yeast (Figure 6). Through restriction endonuclease digestion and linkage, we constructed the An_phy33 display plasmid: pYES2-Hyg-GAL1p-α-An_phy33-SED1-RPL41Bt.
Figure 6 Expression frame of the An_phy33 display plasmid
Build 2:
To construct the plasmid pYES2-Hyg-GAL1p-α-An_phy33-SED1-RPL41Bt, we used a synthetic plasmid containing the An_phy33 sequence as a template to amplify An_phy33 sequence by PCR. 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.
The plasmid was transformed into the E. coli DH5α competent cells. Transformants were successfully cultured overnight, and colony PCR showed the expected 1380 bp band, confirming plasmid transformation.
Figure 7 Construction of the An_phy33 display plasmid
The plasmid was extracted from the positive transformant and linearized. It was then transformed into BY4741 yeast-competent cells and incubated at 30 °C for 48 h. Colony PCR and sequencing verified the correct integration and sequence of the An_phy33 fragment in yeast.
Figure 8 Results of An_phy33 display plasmid transformation of yeast cells.
Test 2:
A positive yeast transformant was cultured to a logarithmic growth period. Galactose induction yielded cells displaying phytase on the surface. Then, the cells were pelleted, washed with 50 mM sodium acetate buffer (pH 5.0), and lysed. Lysate centrifugation gave a supernatant and precipitate containing cell wall-bound phytase. The SDS-PAGE result showed a successful surface display of the An_phy33 phytase (Figure 9).
Phytase activity assays were performed on surface-displayed and cell wall-bound phytase. Lysed phytase precipitate exhibited higher activity and was used to determine phytase activity under different pH and temperature conditions. As shown in Figure 9, the activity of the An_phy33 phytase is higher than that of the AppA phytase. It reaches maximum activity at a pH value of 6 or a reaction temperature of 55 °C.
Figure 9 Expression and enzyme activity test results of An_phy33.
Learn 2:
From the experimental results, it is clear that the An_phy33 phytase had higher activity than the AppA phytase, as predicted. In addition, the optimal reaction pH for AppA was 5, while An_phy33 was 6; and they both had the highest enzyme activities at 55 °C.
Since this technology is currently only performed at the laboratory level and has not been tested in actual feeds, it may result in uncontrollable enzyme activity as a result of temperature and pH. Therefore, its practical application needs to be tested in subsequent experimental validation.