Construction Of Bacillus Subtilis Strain Engineered For Expression Of Porcine β-Defensin-2/cecropin P1 Antimicrobial Peptides And Its Characterization

Bacillus subtilis is a classical probiotic in animal gastrointestinal tract, and the porcine β-defensin-2(pBD-2) and cecropin P1 are crucial antimicrobial peptides(AMPs)in porcine gastrointestinal tract. The probiotics and antimicrobial peptides play the crucial roles in microbiota maintenance, immune regulation and pathogen defending in the tract.Generating probiotics strain engineered for expressing AMPs should be beneficial for animal growth, production and disease resistance. The objective of this study was to express pBD-2 and cecropin P1 in Bacillus subtilis to promote the growth and enhancing disease resistance of piglets. Here, we generated B. subtilis strain engineered for expressing porcine β-defensin-2(pBD-2) and cecropin P1(CP1) fusion antimicrobial peptide. In order to increase the expression level of antimicrobial peptides, the codons for pBD-2 and cecropin P1 genes were optimized using B. subtilis main codons, and the synthesized genes were inserted into pMK4 vector to construct pBD-2/cecropin P1 expression vector pMK4-BD/CP/His, in which the pBD-2 and cecropin P1 genes were fused and were controlled by Lac promoter. Bacteriostatic assay was used to evaluate the bioactivity of the expressed recombinant antimicrobial peptide. Results showed that the B. subtilis strain could efficiently express pBD-2/CP1 fusion antimicrobial peptide,without significantly influencing the growth of engineered B. subtilis by itself-produced fusion peptide. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria(Escherichia coli, Salmonella typhimurium and Haemophilus parasuis) and gram-positive bacteria(Staphylococcus aureus). Supplementing the engineered B.subtilis to the pig feed could significantly promote the growth and the resistance to experimental colibacillosis in weaned piglets. This research provides important scientific evidence for preparation of other probiotic strain expressing recombinant antimicrobial peptides.