Background- Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a disease listed by the world organization for animal health (WOAH) threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and their efficacy is questioned. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. Results- In this study, synthetic biology techniques, recently developed for closely related mycoplasmas, were adapted to Mccp. CReasPy-cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock-out an S41 serine protease gene identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. This approach was further extended to two other field strains of Mccp using CReasPy-Fusion, a method that allows to clone and edit bacterial genomes in yeast through cell-to-cell contact. Furthermore, the Cre-lox recombination system was applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay. Conclusion- Synthetic biology tools were successfully implemented in Mccp. This innovation allows constructing targeted Mccp mutants at ease, which will be of great help to decipher Mccp pathogenicity determinants and develop novel vaccines.< Réduire