Heterologous extracellular production of enterocin P in Lactococcus lactis by a food-grade expression system

In order to develop an entirely food-grade enterocin P expression system for the food industry, the enterocin P structural gene (entP) with or without the enterocin P immunity gene (entiP) was cloned in plasmid pLEB590 under control of the lactococcal constitutive promoter P₄₅. Introduction of the recombinant vectors in L. lactis MG1614 resulted in production of biologically active enterocin P in the supernatants of recombinant L. lactis MG1614. Moreover, coexpression of the entP and entiP genes could increase the production of enterocin P in all L. lactis MG1614 hosts. Recombinant enterocin P from L. lactis MG1614 (pLEB590-entP2) was purified by a three-step procedure involving ammonium sulfate precipitation, SP-Sepharose Fast Flow cation exchange, and hydrophobic adsorption chromatography. The purified bacteriocin protein concentration from recombinant L. lactis MG1614 (pLEB590-entP2) was 3.9-fold greater than that of E. faecium LM-2, and the final recovery of enterocin P activity from the supernatant of L. lactis MG1614 (40.2%) was dramatically improved compared with that of the native host strain (19.9%). Bacteriocin activity and Tricine-SDS–PAGE analysis revealed that purified recombinant enterocin P is biologically active and has a molecular mass corresponding to the native enterocin P from E. faecium LM-2, suggesting that the synthesis, process, and secretion of enterocin P progresses efficiently in recombinant L. lactis MG1614 hosts. The enterocin P was expressed successfully in this food-grade system.