Programmable histone acetylation enables reversible regulation of cellulase production in Myceliophthora thermophila

Myceliophthora thermophila is a thermophilic filamentous fungus with great potential for biomass biorefining and industrial protein production. However, the locus-specific epigenetic regulation of the cellulolytic network in this fungus remains poorly understood. Here, we established a CRISPR/dCas9-p300 system that recruits the p300 histone acetyltransferase to target loci and targeted the promoters of the key cellulolytic regulators clr-1, clr-2, and xyr-1. Targeted recruitment of dCas9-p300 increased the transcription of these regulators by up to 5.2-fold and promoter-associated histone acetylation by approximately 2- to 3-fold. Simultaneous targeting of all three regulators resulted in greater increases than single-locus targeting, with extracellular protein production and CMCase activity increasing by up to 5.5- and 3.7-fold, respectively. The dependence of these effects on guide RNA-directed targeting and p300 catalytic activity was confirmed using no-guide controls and the p300 inhibitor A-485. A catalytically inactive p300 mutant targeting clr-2 failed to enhance clr-2 transcription, extracellular protein production, or CMCase activity. We further developed an inducible dCas9-p300 system to examine the persistence and reversibility of the activated state. Following inducer withdrawal, the high cellulase-production phenotype was maintained during the initial propagation but gradually declined over successive passages and was restored upon re-induction. These results demonstrate that targeted histone acetylation can coordinately activate an endogenous cellulolytic regulatory network and establish a reversible high-production state without altering the DNA sequences of the target loci. This work provides a chromatin-based strategy for reversible regulation of endogenous gene networks and protein production in filamentous fungi.