Multiple copies of the target gene enhances plectasin secretion in Pichia pastoris X-33

Plectasin, the first fungus-derived defensin from Pseudoplectania nigrella, is considered to be one of the most likely substitutes for antibiotics used to cure Staphyloccocus and Streptococcus infections. Recombinant expression of plectasin in Pichia pastoris X-33 was achieved. In this work, we aimed to develop a multi-copy gene cassette to enhance plectasin expression in P. pastoris. Expression vectors pPICZαA-Pn (n = 1, 2, 4, or 8) harboring a one, two, four, or eight-copy gene cassette were constructed and confirmed by digestion with BamHI and BglII. Vectors linearized by BglII were transformed into P. pastoris by electroporation. The gene copy number of the recombinants was quantified by real-time quantitative PCR. Yields of recombinant plectasin were 296, 409, 516, and 879 mg/L for the 1-, 2-, 4-, and 8-copy transformants, respectively, in a 5-L fermentor, demonstrating that an increase in copy number results in a proportional elevation in the expression level of recombinant plectasin. The purity of plectasin was 90.8%, and the yield was found to be 814 mg/L using ion-exchange chromatography. Multi-copy gene dosage enhances plectasin expression levels and highly reduces its production cost. Additionally, the above results could be helpful in improving the application and understanding of the P. pastoris expression system.