ABSTRACT
Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA, from a phylogenetically distant bacterium, Salmonella typhimurium, that drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tunable transient Genetically Modified Organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The manuscript has been extensively revised and rewritten based on feedback from multiple sources. It's quality has been considerably strengthened.
