Enhanced recombinant production of the antimicrobial peptide CRAMP-34 in Pichia pastoris through fermentation optimization and characterization of its antibacterial and stability properties

Antimicrobial peptides are promising alternatives to conventional antibiotics, but their application is limited by inefficient recombinant production. This study established an efficient Pichia pastoris platform for producing mouse-derived CRAMP-34 and evaluated its antibacterial properties. The codon-optimized CRAMP-34 gene was integrated into pPIC9K, and a high-copy P. pastoris GS115 transformant was obtained by G418 screening. Induction conditions were optimized through single-factor experiments and L9 orthogonal design, reaching a maximum freeze-dried product yield of 5.66% at 24°C with 3.0% methanol added every 24h for 144h; the optimum was confirmed in parallel experiments. Recombinant CRAMP-34 demonstrated strong antibacterial activity against Escherichia coli , accompanied by inhibition of biofilm formation and disruption of preformed biofilms. Concentration-dependent increases in bacterial membrane permeability and plasmid DNA binding were observed, indicating that membrane disruption and DNA interaction contributed to the antibacterial activity of CRAMP-34. CRAMP-34 also showed good tolerance to heat, salts, freeze-thaw cycles, and pepsin digestion, although its activity was reduced by trypsin and high-dose papain. These findings establish an efficient production strategy and support CRAMP-34 as a promising antimicrobial agent.