Nuclear ubiquitin-conjugating enzyme TrUbc4 and F-box protein TrFwd1-mediated modification of Cre1 in Trichoderma reesei establishes a regulatory mechanism for carbon catabolite repression

Carbon catabolite repression (CCR) mediated by the transcriptional repressor Cre1 represents a major mechanism ensuring the energy-efficient cellulase production in the model cellulolytic fungus Trichoderma reesei. However, largely unknown is the regulatory pathway governing CCR. In this study, we identified a nuclear ubiquitination system targeting Cre1 to facilitate the induced cellulase gene expression. Either repression of Trubc4 encoding an E2 (ubiquitin-conjugating enzyme) or deletion of Trfwd1 encoding an F-box protein significantly compromised the induced cellulase biosynthesis. However, combinatorial deletion of cre1 suppressed the phenotypic defects resultant from mutations of Trubc4 or Trfwd1. Further analyses demonstrated that TrUbc4 and TrFwd1 collaboratively mediated the ubiquitination of Cre1. Impaired ubiquitination of Cre1 at K361 resulted in its enhanced binding to cellulase gene promoters even under cellulose inducing conditions. This persistent Cre1 binding in turn competitively excluded the functional promoter occupancy of the transcriptional activator Xyr1 required for full cellulase gene expression. These results thus support that Cre1 ubiquitination constitutes a primary mechanism to relieve CCR to ensure the efficient cellulase induction. The present work also highlights the importance of protein ubiquitination for control of carbohydrate utilization and biotechnologically relevant enzyme production in industrial filamentous fungi including Trichoderma reesei.