Efficient extracellular expression of transpeptidase sortase A in Pichia pastoris

- First report on extracellular SrtA expression in Pichia pastoris.
- SMD1168 strain integrating 5.7 copies of the srtA gene with the AOX1 promoter was selected.
- Fermentation conditions for extracellular SrtA expression were optimized.
- Yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL.
- Transpeptidase activity of extracellular expressed SrtA was verified by immobilization of EGFP onto Gly3-polystyrene beads.

In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 °C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly3-polystyrene beads.