Abstract
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 Δstk-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 Δstk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in Δstk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the Δstk-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.
Highlights
Fine-tuning of gene expression is essential for all organisms
We systematically screened N. crassa serinethreonine protein kinase mutants by determining their cellulase production capacity and identified STK-12 to be a crucial factor for cellulase gene downregulation
To identify critical factors involved in turning down the expression of cellulase genes, the deletion mutants of the 64 genes in the N. crassa genome that encode serine-threonine protein kinases were screened through batch culturing with crystalline cellulose (Avicel PH-101) as the sole carbon source
Summary
Fine-tuning of gene expression is essential for all organisms. Complex regulatory networks ensure that the magnitude and duration of gene expression are accurately controlled, and that gene expression is turned off in a timely manner. Defects in the regulation of gene expression are disadvantageous for cell survival [3]. Cellulolytic fungi, such as Trichoderma reesei and Neurospora crassa, play important roles in ecosystems, and are responsible for a major part of plant biomass degradation [4,5]. Previous transcriptome profiling studies have demonstrated that genes encoding major cellulases exhibit a typical bell-shaped expression pattern, implying that fungi produce cellulolytic enzymes in a strictly controlled manner [4]. Previous studies have shown that, when N. crassa is cultured in an inducing medium, the expression levels of cellulase genes dramatically increase at early time points and rapidly decline [4,13]. The transcriptional down-regulation of genes encoding secreted enzymes has been
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call