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Plectasin is a promising and potent antimicrobial peptide isolated from the fungus Pseudoplectania nigrella which has been heterologously expressed in various hosts. In this study, a four-copy cassette of plectasin was constructed via 2A peptide assembly to further increase its expression level in recombinant Pichia pastoris. The yeast transformant 4Ple-61 harboring four-copy cassette of plectasin could secrete 183.2 mg/L total protein containing 60.8% of plectasin at the flask level within 120 h, which was 2.3 times higher than that of the yeast transformant Ple-6 carrying one-copy cassette of plectasin. Western blot confirmed the significant peptide expression level in the transformant 4Ple-61. Furthermore, it yielded as high as 426.3 mg/L total protein within 120 h during a 5-L fermentation. The purified plectasin shows superior stability and good antimicrobial activity against conventional Staphylococcus aureus ATCC 26,001 and some food-borne antibiotic-resistant S. aureus strains with the MICs ranging from 8 to 32 μg/mL. Therefore, the strategy based on 2A peptide assembly can enhance the expression of plectasin and further expand its application prospect. Key points: • A yeast transformant 4Ple-61 with four-copy cassette of plectasin was constructed. • The plectasin level yield by the transformant 4Ple-61 was boosted by 2.3 times. • The plectasin showed good activity against food-borne antibiotic-resistant S. aureus. [ABSTRACT FROM AUTHOR]