Controlled Transcription of Regulator Gene by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in .

Carotenoid biosynthesis is a frequent trait in fungi. In the ascomycete , the synthesis of the carboxylic xanthophyll neurosporaxanthin (NX) is stimulated by light. However, the mutants of the gene, encoding a protein of the RING finger family, accumulate large NX amounts regardless of illumination, indicating the role of CarS as a negative regulator. To confirm CarS function, we used the Tet-on system to control expression in this fungus. The system was first set up with a reporter gene, which showed a positive correlation between the inducer doxycycline and luminescence. Once the system was improved, the gene was expressed using Tet-on in the wild strain and in a mutant. In both cases, increased transcription provoked a downregulation of the structural genes of the pathway and albino phenotypes even under light. Similarly, when the gene was constitutively overexpressed under the control of a promoter, total downregulation of the NX pathway was observed. The results confirmed the role of CarS as a repressor of carotenogenesis in and revealed that its expression must be regulated in the wild strain to allow appropriate NX biosynthesis in response to illumination.