Construction and optimization of secretory genetic manipulation tools in Pichia pastoris

In order to solve the problems of low efficiency and cumbersome process of Pichia pastoris genetic manipulation, secretory genetic manipulation tools were constructed and optimized to improve their industrial production and application value. Firstly, the α-amylase was employed as the reporter protein, and the non-integrated expression plasmid was constructed by fusing the self-replicating sequence. Secondly, endogenous signal peptides that best match the target protein were quickly screened and evaluated using universal primers and POE-PCR. Finally, a nutrition-dependent screening marker was constructed with phosphite as the sole phosphorus source. The results indicate that non-integrated plasmids express α-amylase with 2-fold and 1.6-fold higher enzyme activity compared to integrated plasmids, respectively; The use of FLO10, EXG1, and MSB2 signal peptides results in 2.5-fold, 1.94-fold, and 1.75-fold higher α-amylase activity than the conventional signal peptide α-MF, respectively; Arecombinant strain for heterologous expression of α-amylase is successfully screened using the phosphite dehydrogenase gene (ptxD). The process of gene operation is simplified, the genetic operation tools are improved, and green, safe, and low-cost nutrition-dependent screening markers are provided, which lays a foundation for the application and promotion of the Pichia pastoris secretory expression system.