Extracellular production of antifungal peptides from oxidative endotoxin-free E. coli and application

Antifungal peptides (AFPs) can be used as novel preservatives, but achieving large-scale production and application remains a long-term challenge. In this study, we developed a hybrid peptide MD (metchnikowin-drosomycin fusion) secreted into Escherichia coli supernatant, demonstrating strong inhibitory activity against Aspergillus flavus and Botrytis cinerea. The fusion tag did not impact its activity. Moreover, an endotoxin-free and oxidative leaky strain was developed by knocking out the trxB, gor, and lpp genes of endotoxin-free E. coli ClearColi-BL21(DE3). This strain facilitates the proper folding of multi-disulfide bond proteins and promotes the extracellular production of recombinant bioactive AFP MD, achieving efficient production of endotoxin-free MD. In addition, temperature control replaces chemical inducers to further reduce production costs and circumvent the toxicity of inducers. This extracellularly produced MD exhibited favorable effectiveness in inhibiting fruit mold growth, and its safety was preliminarily established by gavage testing in mice, suggesting that it can be developed into a green and sustainable fruit fungicide. In conclusion, this study provides novel approaches and systematic concepts for producing extracellularly active proteins or peptides with industrial significance. KEY POINTS: • First report of extracellular production of bioactive antifungal peptide in Escherichia coli. • The hybrid antifungal peptide MD showed strong inhibitory activity against Aspergillus flavus and Botrytis cinerea, and the activity was not affected by the fusion tag. • Endotoxin-free oxidative Escherichia coli suitable for the expression of multi-disulfide bond proteins was constructed.