A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

Abstract

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB 590, was constructed entirely of lactococcal DNA: the pSH 71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG 1614 with 60 international units (IU) nisin/ml selection yielded approximately 10(5) transformants/ micro g DNA. MG 1614 carrying pLEB 590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB 590 was successfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.