Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium limosum

Abstract

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.

Development of a recombineering system for the acetogenEubacterium limosumwith Cas9 counterselection for markerless genome engineering

“…Amongst acetogens, E. limosum possesses a growing engineering toolbox including an analyzed genome and transcriptome, an expanded range of selection markers, and libraries of genetic elements, all of which have been used primarily in plasmid-based engineering applications [5], [10]–[13]. Recently, more additions have been made to the organism’s genome engineering repertoire including a CRISPR/dCas9 system for gene silencing and a toxin/antitoxin system for markerless knockout generation [14], [15]. However, the organism’s engineering toolbox could benefit from rapid genome engineering techniques such as homologous recombination to further leverage its native strengths.…”

Development of a Recombineering System for the Acetogen Eubacterium limosum with Cas9 Counterselection for Markerless Genome Engineering

“…Additionally, as a member of the gut microbiota, it has been found to demethylate a range of dietary compounds of relevance to human health, including flavonoids and carnitine, making it an organism of interest for studying microbiota-related pathologies. , Among acetogens, E. limosum possesses a growing engineering toolbox including an analyzed genome, an expanded range of selection markers, and libraries of genetic elements, all of which have been used primarily in plasmid-based engineering applications. ,− Recently, more additions have been made to the organism’s genome engineering repertoire including a CRISPR/dCas9 system for gene silencing and a toxin/antitoxin system for markerless knockout generation. , However, the organism’s engineering toolbox could benefit from rapid genome engineering techniques such as homologous recombination to further leverage its native strengths. We recently reported utilizing the microbe’s native homologous recombination machinery to perform genomic knock-ins and targeted gene deletions .…”