CONICET | Buscador de Institutos y Recursos Humanos

T. thermophila does not require sterols, but synthesizes as a surrogatethe triterpenoid ?tetrahymanol?. If sterols are available, theyare avidly incorporated by the ciliate and bioconverted to 7,22-bisdehydrocholestrol,whereas tetrahymanol synthesis is completelyinhibited. This bioconversion requires four desaturating activities;three of them were previously characterized by our groups. Herewe describe the identification of the last fourth enzyme, the C22 desaturase,instrumental in the development of microbial systems forthe synthesis of tailored steroids. All known C22 sterol desaturasesbelong to the P450 oxygenases, essential enzymes for the synthesisof ergosterol in fungi or phytosterols in plants. T. thermophilahas 44 putative P450 oxygenases, all of them sharing low similarityto canonical C22 desaturases. The ciliate enzyme was induced bysterols, required NAD(P)H and was inhibited by azide and cyanide,but not by azoles, typical properties of oxygenases belonging to thesuperfamily fatty-acid desaturases/hydroxylases (FAD/H). Analysis of a differential transcriptome carried out on T. thermophila, grown inpresence or absence of sterols, allowed us to detect several FAD/Hsignificantly induced by sterols. The two more induced ones, namedDes1 and Des2 (sharing 68% similarity) were selected for furthercharacterization. Expression of Des1 or Des2 in a Saccharomycescerevisiae C22 desaturase mutant, both rescued the synthesis ofthe yeast ergosterol. Additional genetic approaches performed onthe ciliate have unambiguously confirmed the C-22 activity of thesetwo isoenzymes. It is the first description of a C22 sterol desaturasewhich is not a P450 oxygenase. These results also expand the repertoireof FAD/H specificities.Keywords: sterols, bioconversion, desaturases, ciliates

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