CRISPR-Cas9-mediated seb1 disruption in Talaromyces pinophilus EMU for its enhanced cellulase production.

Filamentous fungi are working horses for industrial enzyme production. Combinatory approaches, such as random mutagenesis and rational genetic engineering, were adopted to improve their enzyme productivity. The filamentous fungus Talaromyces pinophilus EMU is a hyper cellulase-producing filamentous fungus obtained through random mutagenesis. This study further enhanced its cellulase production through the disruption of seb1 gene, which encodes Seb1, a transcription factor that binds to the stress response element (STRE) and regulates a variety of cellular processes. Gene seb1 was cloned from strain T. pinophilus EMU and disrupted using CRISPR-Cas9 technology. The seb1-disruptants (TpΔseb1 strains) showed distinct morphology from its parent strain. They presented a hyphal branching phenotype with decreased transcription levels of rhoA and ras1 genes involved in hyphal branching. Furthermore, TpΔseb1 strains displayed lower cell biomass, higher specific protein content, and 20%-40% enhancement in filter paper cellulase (FPase) activity, however, insignificant changes in the transcription levels of cbh1 and bgl1 genes involved in cellulase production. Through this study, we confirmed that seb1 gene disruption in T. pinophilus EMU caused more hyphal branching, reduced cell growth, increased protein secretion, and enhanced cellulase production. In addition, we successfully established the CRISPR-Cas9 genome-editing platform in T. pinophilus EMU.