System biology approaches for improving the production of industrial cellulolytic enzymes

Cellulolytic fungi have evolved sophisticated systems for adaptation to their natural habitat. The transcription regulatory network that governs expression of cellulolytic genes remains poorly understood. For improving the cellulases production by an industrial strain of Penicillium oxalicum, a gene disruptant mutant library of 470 transcriptional regulators in P. oxalicum was constructed, and screening for transcription factors associated with cellulose degradation. Twelve transcription factors that play a pivotal role in the activation or repression of cellulase genes were identified. CreA was found as a global regulator of carbon catabolite repression (CCR) in P. oxalicum, and negatively regulate expression of cellulolytic genes. Deletion of creA promotes cellulolytic gene expression under all growth conditions. Another transcription factor ClrB play opposing roles with CreA in cellulolytic gene expression. Overexpression of clrB is sufficient to increase cellulase activity, while its deletion abrogates cellulase gene expression. It was found that clrB overexpression alone is insufficient to drive expression of cellulolytic genes on glucose, but combined with creA deletion, strong transcriptional upregulation of cellulolytic genes and cellulolytic activity were obtained on both inducing and repressing conditions. More combinatorial gene regulation involving several transcription factors were observed in mutation and overexpression experiments. These data refined our understanding of transcriptional-regulatory network as a “seesaw model” in which coordinated regulation of cellulolytic genes is established using activators and repressors counteracting under inducing condition.