Site-directed mutagenesis of Aspergillus niger phytase gene and its cell-surface display on Komagataella phaffii
2018
YU Daobing | CHENG Xuesong | WANG Qun | SHI Yanke | ZHANG Xin
A full-length sequence of phytase gene (phyA) from Aspergillus niger ZJUY was cloned and modified based on polymerase chain reaction (PCR)-directed mutagenesis, aiming at addressing phytase denaturalization and deactivation during the process of pelleting. The mutation types included codon optimization for key sites, the introduction of hydrogen bond (T295S, Q296R and V43N) and the deletion mutation of disulfide bond (Cys196-Cys446). The anchored- Flo1p (FS) and target-phyA were successfully inserted into the yeast expression vector pPICZαC by seamless cloning. Eight kinds of expression vector pPICZαC- FS/phyA were integrated into the Komagataella phaffii GS115 genome by homologous recombination using lithium chloride transformation method, respectively. Phytases were successfully displayed on the surface of K. phaffii GS115, verified by immunofluorescence staining and flow cytometry. The resulted recombinants A31, A61 and A84 all showed a high phytase activity of 7 000 U/g. In addition, phytase-displaying exhibited better thermostability and pH-stability than those of secretory phytase. Among them, A61 remained 18% phytase activity after water bath at 90℃ for 1 h, and still maintained more than 80% of phytase activity at a pH range of 1.6 to 4.0. This characteristic of strain A61 can overcome deactivation during the process of granulating at 60 to 90℃ and better meet the demand in animal feed and food processing.
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