Construction and characterization of thymidine auxotrophic (ΔthyA) recombinant Lactobacillus casei expressing bovine lactoferricin

Abstract

BACKGROUND: Lactobacillus casei (L. casei) is well known for its probiotic property in human and animals. Lactoferricin (Lfcin) polypeptide can effectively modulate host immune responses and have antimicrobial activity in vivo and in vitro. In order to develop a food-grade L. casei system constitutively expressing bovine Lfcin, this study constructed a thymidine auxotrophy (ΔthyA) recombinant L. casei. RESULTS: Based on the thymidylate synthase gene (thyA) insert site, LFEC(Lfcin expression cassette)was inserted into L. casei genome through homologous recombination, successfully expressed and could be stably inherited. The recombinant L. casei, ΔthyA L. casei-LFEC, is sensitive to chloramphenicol and limited when cultured without thymine. Meanwhile, ΔthyA L. casei-LFEC has both good antibacterial activity against Escherichia coli and Staphylococcus aureus and antiviral activity against porcine epidemic diarrhea virus (PEDV). CONCLUSIONS: We successfully constructed a recombinant L. casei strain expressing Lfcin, ΔthyA L. casei-LFEC, which could only survive in the presence of thymine, and had excellent antimicrobial and antiviral activity with good genetic stability and sensitivity. This research provides a cost-effective alternative to the antibiotics with additional biological functions and wider applicability prospect. Using ΔthyA as the selectable mark instead of antibiotic to construct genetic engineering L.casei provides a safe and effective approach of feed additives in livestock raising.