Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475

Abstract

La producción heteróloga de la bacteriocina enterocina A (EntA) se evaluó en diferentes Lactobacillus spp. después de la fusión del versátil péptido señal dependiente de Sec (SPusp45) con la EntA madura más el gen de inmunidad EntA (entA + entiA) (fragmento UAI), y su clonación en vectores plasmídicos que permitieron su producción inducida (pSIP409 y pSIP411)constitutiva (pMG36c ). La cantidad, actividad antimicrobiana (AA) y actividad antimicrobiana específica (SAA) del EntA producido por Lactobacillus sakei Lb790, Lb. plantarum NC8 y Lb. casei CECT475 transformada con los plásmidos recombinantes pSIP409UAI, pSIP411UAI y pMGUAI variaban dependiendo del vector de expresión y la cepa huésped. Las cepas recombinantes casei Lb. casei CECT475 produjeron las cantidades más grandes de EntA, con los más altos niveles de AA y SAA. Sobrenadantes de Lb. casei CECT (pSIP411UAI) mostraron una producción 4,9 veces mayor de EntA con un AA 22,8 veces mayor y un SAA 4,7 veces mayor que los de Enterococcus faecium T136, el productor natural de EntA. Además, los sobrenadantes de Lb. casei CECT475 (pSIP411UAI) mostraron una AA de 15.7 a 59.2 veces mayor contra Listeria spp. que los de E. faecium T136. Lb. Casei CECT457 (pSIP411UAI) puede considerarse una prometedora fábrica de hospedadores y células recombinantes para la producción y expresión funcional de la bacteriocina antiemisterial EntA.

Bacteriocins produced by lactic acid bacteria (LAB) attract considerable interest as natural and nontoxic food preservatives and as therapeutics whereas the bacteriocin-producing LAB are considered potential probiotics for food, human and veterinary applications, and in the animal production field. Within LAB the lactobacilli are increasingly used as starter cultures for food preservation and as probiotics. The lactobacilli are also natural inhabitants of the gastrointestinal (GI) tract and attractive vectors for delivery of therapeutic peptides and proteins, and for production of bioactive peptides. Research efforts for production of bacteriocins in heterologous hosts should be performed if the use of bacteriocins and the LAB bacteriocin-producers is ever to meet the high expectations deposited in these antimicrobial peptides. The recombinant production and functional expression of bacteriocins by lactobacilli would have an additive effect on their probiotic functionality. The heterologous production of the bacteriocin enterocin A (EntA) was evaluated in different Lactobacillus spp. after fusion of the versatile Sec-dependent signal peptide (SP usp45 ) to mature EntA plus the EntA immunity gene (entA + entiA) (fragment UAI), and their cloning into plasmid vectors that permitted their inducible (pSIP409 and pSIP411) or constitutive (pMG36c) production. The amount, antimicrobial activity (AA) and specific antimicrobial activity (SAA) of the EntA produced by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 transformed with the recombinant plasmids pSIP409UAI, pSIP411UAI and pMGUAI varied depending of the expression vector and the host strain. The Lb. casei CECT475 recombinant strains produced the largest amounts of EntA, with the highest AA and SAA. Supernatants from Lb. casei CECT (pSIP411UAI) showed a 4.9-fold higher production of EntA with a 22.8-fold higher AA and 4.7-fold higher SAA than those from Enterococcus faecium T136, the natural producer of EntA. Moreover, supernatants from Lb. casei CECT475 (pSIP411UAI) showed a 15.7- to 59.2-fold higher AA against Listeria spp. than those from E. faecium T136. Lb. casei CECT457 (pSIP411UAI) may be considered a promising recombinant host and cell factory for the production and functional expression of the antilisterial bacteriocin EntA.