show Abstracthide AbstractMycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Using synthetic biology approaches, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri (Mmc) genome cloned in yeast, reproducing mutations known to be associated with a temperature-sensitive phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety.