Overview

To address the shortage of feed protein sources, some alternative proteins are being explored. Single-cell proteins produced by fermentation to generate microbial biomass products for human or animal consumption are rich in amino acids and good substitutes. Common animal feeds require the addition of enzymes like xylanase and phytase to eliminate antinutritional factors from plant ingredients.

In this project, to expand the application of phytase 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. Additionally, we introduced a new basic part, An_phy33, to increase phytase enzymatic activity.

 

 

1. Add new functional data to an existing part BBa_K912001 and a new part BBa_K4870016

To obtain AppA phytase displayed on the yeast cell surface, we inserted the AppA gene into the plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt (BBa_K4870015), where the SED1 protein targets AppA to the yeast cell wall. After transforming the recombinant plasmid into Saccharomyces cerevisiae cells and inducing expression with 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.

 

AppA SDS-PAGE and phytase activity under different (A) pH and (B) temperature conditions.

 

2. Create new parts BBa_K4870001 and BBa_K4870017 with biological information and experimental data

We obtained a series of ancestral AppA sequences from E. coli using the website (https://loschmidt.chemi.muni.cz/fireprotasr/). Prediction using DeepSTABp (https://csb-deepstabp.bio.rptu.de/) showed the An_phy33 sequence had the highest melting temperature Tm of 87°C, compared to 51°C for wild-type AppA. Thus we selected this sequence for our experiments.

To obtain the An_phy33 phytase displayed on the yeast surface, we inserted the An_phy33 gene into the plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, where the SED1 protein targets An_phy33 to the cell wall. After transforming the plasmid into Saccharomyces cerevisiae and inducing it with galactose, we obtained surface-displayed An_phy33 phytase. Enzyme activity was assayed under different pH and temperature conditions. We found that the An_phy33 phytase had higher activity than the AppA phytase, and it reaches maximum activity at a pH value of 6 or a reaction temperature of 55 °C.

 

An_phy33 SDS-PAGE and phytase activity under different (A) pH and (B) temperature conditions.