Reconstitution in yeast of the pro-vitamin D3-synthesis complex from Tetrahymena thermophila

MARÍA CELESTE MOLINA; SEBASTIÁN R. NAJLE; ANTONIO D. UTTARO

Rosario

Jornada; L Reunión Anual de la SAIB; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Using a reverse genetic approach, we have previously identified a Rieske oxigenase as the sterol C7 desaturase of T. thermophila. It is involved in the biotechnologically relevant conversion of cholesterol into 7-dehydrocholesterol or pro-vitamin D3. In order to carry out its biochemical characterization, the oxigenase gene was fully synthesized with the codon usage of yeasts, and expressed in an appropriate Saccharomyces cerevisiae strain. C7-desaturase activity was assayed providing cholesterol to the yeast culture and analyzing the sterol profile by TLC and GC/MS. Reconstitution of an active system, with significant 7-dehydrocholesterol production, was possible only after coexpressing the human NADPH cytochrome P450 oxidoreductase, indicating that yeasts lack a compatible electron donor for the Rieske oxigenase. A BLASTp search revealed the presence of four putative oxidoreductase orthologues in T. thermophila. Applying the same reverse genetics technique, based on the RNA interference by feeding, one of the genes was identified as the natural electron donor in the enzymatic complex. It makes feasible the development of a sustainable system for the industrial production of provitamin D3.