CONICET | Buscador de Institutos y Recursos Humanos

T. thermophila does not require sterols, but synthesizes as a surrogate the triterpenoid ?tetrahymanol?. If sterols are available, they are avidly incorporated by the ciliate and bioconverted to 7, 22-bisdehydrocholestrol, whereas tetrahymanol synthesis is completely inhibited. This bioconversion requires four desaturating activities; three of them were previously characterized by our groups. Here we describe the identification of the last fourth enzyme, the C22 desaturase, instrumental in the development of microbial systems for the synthesis of tailored steroids. All known C22 sterol desaturases belong to the P450 oxygenases, essential enzymes for the synthesis of ergosterol in fungi or phytosterols in plants. T. thermophila has 44 putative P450 oxygenases, all of them sharing low similarity to canonical C22 desaturases. The ciliate enzyme requires NAD(P)H and was inhibited by azide and cyanide, but not by azoles, typical properties of oxygenases belonging to the superfamily fatty-acid desaturases/hydroxylases (FAD/H). Analysis of a differential transcriptome carried out on T. thermophila, grown in presence or absence of sterols, allowed us to detect several FAD/H significantly induced by sterols. The two more induced ones, named Des1 and Des2 (sharing 68% similarity) were selected for further characterization. Expression of Des1 or Des2 in a Saccharomyces cerevisiae C22 desaturase mutant, both rescued the synthesis of the yeast ergosterol. Molecular approaches performed on the ciliate have unambiguously confirmed the C-22 activity of these two isoenzymes. It is the first description of a C22 sterol desaturase which is not a P450 oxygenase, and also expand the repertoire of FAD/H specificities.Keywords: sterols, bioconversion, desaturases, ciliates