A simple strategy for the generation of multi-copy Pichia pastoris with the efficient expression of mannanase

Pichia pastoris expression system was widely used for producing heterologous proteins. Some researches revealed that the increase in the copy number could improve the expression of foreign genes in P. pastoris. Hundreds or thousands of antibiotic-resistance recombinants need to be screened because the frequency of multiple gene insertion events is very low in P. pastoris. The traditional method of constructing multi-copy gene is complicated, tedious, and full of randomness. Here, we developed a rapid method for constructing multi-copy Pichia expression vectors harboring mannanase gene. The developed strategy is easy to manipulate genetically and can precisely generate plasmids with a certain copy number of heterologous gene. The average mannanase activities of recombinants randomly chosen from two-, four-, and six-copy recombinant libraries were 1.7-, 2.2-, and 1.3-folds, respectively, of that from single-copy recombinant library. The result revealed that the strategy could effectively improve the expression of foreign proteins in P. pastoris.