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

• Published in: Biotechnology journal Vol. 14; no. 8; p. e1800587
• Main Authors: Duarte, Sofia O D, Martins, Maria C, Andrade, Sílvia M, Prazeres, Duarte M F, Monteiro, Gabriel A
• Format: Journal Article
• Language: English
• Published: Germany 01.08.2019
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Computer Simulation; DNA Copy Number Variations; Genetic Engineering - methods; Lactococcus lactis - genetics; Lactococcus lactis - growth & development; Mutagenesis, Site-Directed; Plasmids; Real-Time Polymerase Chain Reaction; Ribosomes - metabolism; RNA, Messenger - analysis; RNA, Messenger - chemistry; site-directed mutagenesis; Lactococcus lactis; ribosome-binding sites; pTRKH3; plasmid copy numbers
• ISSN: 1860-7314
• DOI: 10.1002/biot.201800587

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.