High-efficiency expression of a novel antimicrobial peptide I20 with superior bactericidal ability and biocompatibility in Pichia pastoris and its efficiency enhancement to aquaculture

One of the major constraints on aquaculture is the susceptibility of farmed fish to diseases or slow growing. Antimicrobial peptide I20, as a novel feed additive, is used to cope with these limiting factors. In the current study, the Pichia pastoris expression system was used to produce I20 in large quantities, thereby reducing its application cost. In terms of molecular strategy, the I20 expression codon was optimized according to the preference of P. pastoris codon. Through traditional strategy optimization (expression strain, time, methanol concentration, temperature, and pH), the expression level of I20 in shaking bottles was 21.09 +/- 1.10 mg/L. Surprisingly, the expression level of I20 in a 20 L high-cell-density cultivation was 514.78 +/- 22.07 mg/L after methanol induction of 72 h. Comprehensive analyses using a series of fluorescence and microscopy revealed that I20 killed bacteria by increasing bacterial membrane permeability and binding bacterial DNA. I20 effectively killed a variety of pathogenic bacteria (Aeromonas hydrophila, Aeromonas veronii, Elizabethkingia miricola, Fla-vobacterium columnare, Edwardsiella ictaluri, Vibrio parahaemolyticus, Streptococcus agalactiae, Nocardia seriolae, Escherichia coli, and Staphylococcus aureus). I20 was found to have excellent biocompatibility after both feeding and injection studies. Grass carp were fed I20 diet supplemented with 200 mg/Kg I20 for 8 weeks. I20 can effectively promote intestinal villus length, secretion of digestive enzymes, and expression of growth genes to improve growth performance. I20 significantly promotes immune response to reduce tissue damage and bacterial load, enhancing survival after Aeromonas hydrophila infection. In conclusion, we systematically optimized the expression of antimicrobial peptide I20 in P. pastoris, and achieved a satisfactory efficiency for producing antimicrobial peptide I20 by integrating several strategies. The results herein showed that I20-feed promoted growth performance and disease resistance in fish, suggesting a feed supplement with great potential applica-tions for aquaculture.