Our project is centered on the construction and optimization of efficient expression systems in Pichia pastoris, with a particular emphasis on contributing well-characterized biological parts to the iGEM community within the domain of synthetic biology. We selected a comprehensive set of high-expression regulatory elements, including promoters AOX1, FDH1, CAT1, and AOX713, as well as signal peptides α-factor, SP4, SP14, and 0030. These components represent either native or engineered promoters and signal peptides of Pichia pastoris, each demonstrated to enhance the expression and secretion efficiency of heterologous proteins. To facilitate the industrial-scale production of the antimicrobial peptide Plectasin NZ2114, we systematically integrated these regulatory elements into the pPIC9K plasmid vector, constructed multiple recombinant expression strains, and performed functional validation, resulting in improved expression levels and bioactivity of Plectasin NZ2114 in Pichia pastoris.

Antimicrobial peptides have emerged as promising alternatives to conventional antibiotics due to their broad-spectrum activity and low propensity for inducing resistance. Heterologous protein expression via genetic engineering offers a viable solution to overcome challenges related to low yield and high production costs. As a safe and highly efficient host for recombinant protein expression, Pichia pastoris relies heavily on the selection of appropriate promoters and signal peptides to leverage its robust secretory pathway. Through systematic evaluation and optimization of these expression components, our work not only advanced the development of high-producing Plectasin NZ2114 strains but also established a validated, modular, and reusable toolkit of expression parts—readily applicable by the iGEM community and other researchers for the scalable production of antimicrobial peptides and other recombinant proteins.

In summary, this study significantly expands the repertoire of functional parts available for Pichia pastoris expression systems. Furthermore, it provides valuable insights and a practical foundation for the rational design of expression constructs in synthetic biology and metabolic engineering. The developed platform holds strong potential to drive future technological advancements and broaden the industrial applications of microbial cell factories.