Plasmid Copy Number of pTRKH3 in Lactococcus lactis is Increased by Modification of the repDE Ribosome‐Binding Site

摘要

> Plasmids for DNA vaccination are exclusively produced in the Gram‐negative Escherichia coli . One important drawback of this system is the presence of lipopolysaccharides. The generally recognized as safe Lactococcus lactis ( L. lactis ) would constitute a safer alternative for plasmid production. A key requirement for the establishment of a cost‐effective L. lactis ‐based plasmid manufacturing is the availability of high‐copy number plasmids. Unfortunately, the highest copy number reported in Gram‐positive bacteria for the pAMβ1 replicon is around 100 copies. The purpose of this work is to engineer the repDE ribosome‐binding site (RBS) of the pTRKH3 plasmid by site‐directed mutagenesis in order to increase the plasmid copy number in L. lactis LMG19460 cells. The pTRKH3‐b mutant is the most promising candidate, achieving 215 copies of plasmid per chromosome, a 3.5‐fold increase when compared to the nonmodified pTRKH3, probably due to a stronger RBS sequence, a messenger RNA secondary structure that promotes the RepDE expression, an ideal intermediate amount of transcriptional repressors and the presence of a duplicated region that added an additional RBS sequence and one new in‐frame start codon. pTRKH3‐b is a promising high‐copy number shuttle plasmid that will contribute to turn lactic acid bacteria into a safer and economically viable alternative as DNA vaccines producers.