Towards the creation of Acholeplasma laidlawii driven by synthetic genomes

Abstract

ABSTRACTAssembling synthetic bacterial genomes in yeast and genome transplantation has enabled an unmatched level of bacterial strain engineering, giving rise to cells with minimal and chemically synthetic genomes. However, this technology is currently limited to members of the Spiroplasma phylogenetic group, mostly Mycoplasmas, within the Mollicute class. Here, we propose the development of these technologies for Acholeplasma laidlawii, which is phylogenetically distant from Mycoplasmas and, unlike most Mollicutes, uses a standard genetic code. Towards this goal, we first investigated a donor-recipient relationship between two A. laidlawii strains through whole-genome sequencing. We then created a multi-host shuttle plasmid and used it to optimize an electroporation protocol. For genome transplantation, we selected A. laidlawii 8195 as the recipient strain and we created a PG-8A donor strain by inserting a Tn5 transposon carrying a tetracycline resistance gene. Our optimized genetic tools will accelerate the creation of Acholeplasma strains driven by synthetic genomes.Graphical Abstract