Single Crossover-Mediated Markerless Genome Engineering in Clostridium acetobutylicum

Abstract

A novel genome-engineering tool in Clostridium acetobutylicum was developed based on single-crossover homologous recombination. A small-sized non-replicable plasmid, pHKO1, was designed for efficient integration into the C. acetobutylicum genome. The integrated pHKO1 plasmid backbone, which included an antibiotic resistance gene, can be excised in vivo by Flp recombinase, leaving a single flippase recognition target sequence in the middle of the targeted gene. Since the pSHL-FLP plasmid, the carrier of the Flp recombinase gene, employed the segregationally unstable pAM beta 1 replicon, the plasmid was rapidly cured from the mutant C. acetobutylicum. Consequently, our method makes it easier to engineer C. acetobutylicum.