STK-12 acts as a transcriptional brake to control the expression of cellulase-encoding genes in Neurospora crassa

Cellulolytic fungi have evolved a complex regulatory network to maintain the precise balance of nutrients required for growth and hydrolytic enzyme production. When fungi are exposed to cellulose, the transcript levels of cellulase genes rapidly increase and then decline. However, the mechanisms underlying this bell-shaped expression pattern are unclear. We systematically screened a protein kinase deletion set in the filamentous fungus Neurospora crassa to search for mutants exhibiting aberrant expression patterns of cellulase genes. We observed that the loss of stk-12 (NCU07378) caused a dramatic increase in cellulase production and an extended period of high transcript abundance of major cellulase genes. These results suggested that stk-12 plays a critical role as a brake to turn down the transcription of cellulase genes to repress the overexpression of hydrolytic enzymes and prevent energy wastage. Transcriptional profiling analyses revealed that cellulase gene expression levels were maintained at high levels for 56 h in the Delta stk-12 mutant, compared to only 8 h in the wild-type (WT) strain. After growth on cellulose for 3 days, the transcript levels of cellulase genes in the Delta stk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in Delta stk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the Delta stk-12 mutant was 7-fold higher than in the WT. These results strongly suggest that stk-12 deletion results in dysregulation of the cellulase expression machinery. Further analyses showed that STK-12 directly targets IGO-1 to regulate cellulase production. The TORC1 pathway promoted cellulase production, at least partly, by inhibiting STK-12 function, and STK-12 and CRE-1 functioned in parallel pathways to repress cellulase gene expression. Our results clarify how cellulase genes are repressed at the transcriptional level during cellulose induction, and highlight a new strategy to improve industrial fungal strains. Author summary Microorganisms can sense and respond to nutrient availability in the external environment, and turn on/off cellular signaling pathways to control gene expression in a timely manner. In filamentous fungi, the expression of hydrolytic enzymes is tightly controlled at the transcriptional level. Within fungal cells, signals from induction and repression pathways are integrated, resulting in optimal hydrolase gene expression. However, the detailed molecular mechanism of transcriptional down-regulation of hydrolytic enzyme genes remains poorly understood. The filamentous fungus Neurospora crassa, a native degrader of lignocellulosic biomass, was developed as a model to unravel mechanisms of lignocellulolytic gene regulation. Using this system, we systematically screened N. crassa serine-threonine protein kinase mutants by determining their cellulase production capacity and identified STK-12 to be a crucial factor for cellulase gene downregulation. Since STK-12 is conserved across species, our data potentially provide insights into the repression mechanism that controls hydrolase gene expression also in other filamentous fungi, and will be useful in the rational engineering of fungal strains to improve industrial enzyme production.