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

Abstract

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.