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

Abstract

BackgroundLactobacillus 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.ResultsBased 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).ConclusionsWe 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.

Effects of Lactococcus lactis MG1363 producing fusion proteins of bovine lactoferricin–lactoferrampin on growth, intestinal morphology and immune function in weaned piglet

“…Next, 18% Tricine‐SDS‐PAGE and Western blot assay were performed using an anti‐bLF polyclonal antibody (prepared by our laboratory and diluted at 1 : 500) as the primary antibody and a horseradish peroxidase‐conjugated goat anti‐mouse immunoglobulin G (IgG) (1 : 4000) as the secondary antibody. Moreover, enzyme‐linked immunosorbent assay (ELISA) was used to quantify the concentration of the LFCA protein produced by the pAMJ399‐LFCA/LLMG1363, using the principles previously described (Zhou et al, ).…”

Safety Aspects of Genetically Modified Lactic Acid Bacteria

“…Recombinant Lb. casei ∆thyA that expresses bovine lactoferricin was also successfully constructed, and only survived in the presence of thymine [64]. A similar auxotroph transgene containment method has targeted the pyrG gene, which encodes CTP synthase, and is thus responsible for converting UTP to CTP during de-novo pyrimidine synthesis in L. lactis.…”

“…An expression vector that carries a complementation is constructed to complement for gene deletion in the host chromosome. Systems based on auxotrophic markers (which are often used to achieve biological containment) in LAB mainly depend on complementation of thymine or cytidine [62,64,65]. Several systems have been applied to L. lactis.…”

Developing a broadly applicablepyrF-based genetic manipulation system inAcinetobacter baumannii

“…Auxotrophic makers, such as amino acid auxotrophy, NADPH auxotrophy, thymidine auxotrophy and uracil auxotrophy, are useful tools in gene editing and vaccine products [21][22][23][24][25]. The uracil auxotrophy has been successfully constructed in multiple strains, and some uracil related genes, such as pyrF and ura3, are also widely used [23,[26][27][28].…”