Steroids are strategic drugs for our health system and therefore, FRONTEST aims to innovate their biotechnological production processes currently based on natural sterols using advanced technologies of metabolic engineering and synthetic biology in Mycocilibacterium smegmatis as a cell factory. The first objective is to develop the biosynthesis of C22 side chain steroid synthons that are used in the semisynthesis of progesterone and bile acids. Mutants of M. smegmatis will be constructed by means of a rational design, modifying the genes involved in the degradation of the side chain of sterols. The second objective focuses on optimizing the performance in the production of synthons by reducing the accumulation of contaminating by-products, either by deleting or supplementing genes in a second round of mutagenesis. Through these two objectives, it will also be possible to accurately complete the knowledge of the catabolic pathway of cholesterol degradation of great relevance in the pathogenesis of tuberculosis. The third very innovative, but also risky, objective consists in the study of the cholesterol degradation pathway in Caenebius tardaugens, a unique bacterium, since our preliminary data suggest that sterols are metabolized through the formation of progesterone and therefore, it would be a new degradative pathway not described to date. These studies will also allow us to generate a new methodology for the single step production of progesterone from natural sterols and to develop a priori more efficient production of C19 steroid synthons, designing à la carte a new catabolic pathway for natural sterols with fewer steps than the pathways described to date.