Background: Mycoplasmas are cell wall-less bacteria that have arisen as biological models due to their small genomes. However, their genetic manipulation is hindered by the small number of tools available. Recently, key synthetic biology technologies have allowed the large-scale modification of several mycoplasma genomes. They consist in transferring the bacterial genome into yeast, modifying it using yeast tools and transferred it back into bacterial cells to produce a mutant. Although successful for several ruminant-pathogenic mycoplasmas, this approach remain ineffective for bacteria such as Mycoplasma pneumoniae.Methods: In this study, we developed an alternative strategy that takes advantage of the Recombinase-Associated Genomic Engineering, allowing the transfer of large genomic fragments from yeast to bacteria. First, we use the Transformation-Associated Recombination-cloning (TAR-cloning) method to capture, between heterospecific lox sites, a large fragment of bacterial genomic DNA into a YAC-BAC shuttle vector, where it can be modified using the tools available in yeast. This vector is then transformed in Escherichia coli to allow its amplification and eventual further modification. Last, we use the Recombinase-Mediated Cassette Exchange strategy, to integrate the genomic fragment into M. pneumoniae genome at a specific locus.Results: Using this method, we introduced a 15 kbp DNA fragment in an intended locus into the M. pneumoniae genome and replaced a 38 kbp wild-type genomic region by a quasi-equivalent fragment (natural or synthetic) in which candidate genes were deleted. Conclusion: This new technology enables to perform large-scale genomic modifications at specific loci in an organism considered, until recently, as genetically intractable.< Réduire