Reconstruction of expression regulation network (REXRN) for improving the cellulase production and enzyme system composition of Penicillium oxalicum

Cellulolytic fungi have evolved sophisticated systems for adaptation to their natural habitat. Penicillium oxalicum is a promising cellulase producer. For understanding its expression regulation network, a library of transcription factor mutants was constructed, and more than ten transcription factors were identified concerning with the regulation network of cellulolytic enzymes. It was found that the strains carrying combined deletions of transcription repressors with overexpression of activators have increased their cellulolytic gene expression. Especially, deletion of intracellular beta-glucosidase Bgl2 encoding gene (enhancing the induction by blocking intracellular inducer hydrolysis) was efficient for enhancing the cellulolytic gene expression, combined with overexpression of the main activating regulators, ClrB and XlnR, and deletions of the repressors, CreA and AmyR. Characterization of a trigenic recombinant, named as RE-10, containing deletions of bgl2 and creA and constitutive overexpression of clrB showed coordinated regulation of cellulolytic genes. The cellulolytic ability of RE-10 was significantly improved, even higher than that of the industrial P. oxalicum strain JU-A10-T obtained after 30 year’s random mutagenesis and screening.

The reconstruction of expression regulation network (REXRN) refined our understanding of transcriptional regulatory network as a “seesaw model” in which coordinated regulation of cellulolytic genes using activators and repressors counteracting.

Comparative transcriptomic and proteomic analysis provided further insights into the differential secretomes between RE-10 and wild type strains. In particular, the enzymes and accessory proteins involved in lignocellulose degradation were elevated specifically and dramatically in the recombinant, thereby confirming the importance of them in biomass deconstruction, and implying a possible co-regulatory mechanism.