Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli

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.

Gibson Assembly-based direct cloning of plasmid DNA in Lactiplantibacillus plantarum WCSF1

“…To demonstrate this, a simple plasmid construct, pLp_mCherry, with gene sequences ideal for indirect cloning through E. coli was used. This plasmid consisted of a p256 replicon, an erythromycin resistance cassette and the mCherry gene driven by a strong constitutive promoter P tlpA , [34] all of which are compatible in both E. coli and L. plantarum WCFS1. The plasmid was constructed through Gibson assembly of the pLp-3050sNuc plasmid backbone and P tlpA -mCherry insert and transformed in E. coli .…”