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

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.

The Secretome of Phanerochaete chrysosporium and Trametes versicolor Grown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials

“…This behavior suggested an initial induction period as already demonstrated for P. chrysosporium K-3 strain (Suzuki et al, 2009), whereas the late decrease in cbh transcripts is likely associated with repression caused by glucose released from Avicel at advanced culture periods (Daly et al, 2019). However, total FPA progressively accumulated in the culture media along the 12 days culture period ( Supplementary Figure S1), indicating a delay between gene transcription and enzyme secretion and extracellular accumulation (Midorikawa et al, 2018;Lin et al, 2019).…”

“…Lignocellulosic substrates or pure polysaccharides and their degradation products have been used to induce secretion of plant biomass degrading proteins in fungi ( Szabó et al, 1996 ; Suto and Tomita, 2001 ; Ahamed and Vermette, 2009 ; Juturu and Wu, 2014 ; Payne et al, 2015 ; Bischof et al, 2016 ; Presley and Schilling, 2017 ; Lin et al, 2019 ). Certain studies of white-rot fungi have shown that Avicel (a commercial preparation of microcrystalline cellulose) and its biodegradation products act as GH inducers in the model basidiomycete Phanerochaete chrysosporium ( Uzcategui et al, 1991 ; Broda et al, 1995 ; Szabó et al, 1996 ; Suzuki et al, 2010 ).…”

High-dose rapamycin exerts a temporary impact on T. reesei RUT-C30 through gene trFKBP12

“…Gene sch9 is a critical component of the TOR signaling pathway in Saccharomyces cerevisiae . Deletion of its homolog stk-10 in N. crassa severely decreased cellulase production [ 10 ]. Similarly, loss of schA (the homolog of stk-10 ) in Aspergillus nidulans caused defects in protein production and cellulolytic enzyme activities [ 11 ].…”

Parallel proteomic and phosphoproteomic analyses reveal cellobiose‐dependent regulation of lignocellulase secretion in the filamentous fungus Neurospora crassa

“…Altogether, the results suggest that cellobiose regulates the early adaptation of N . crassa to cellulose at post‐transcriptional, translational, and post‐translational levels (Collier et al, 2020; Cziferszky et al, 2002; Li, Pang, et al, 2019; Lin et al, 2019; Liu et al, 2020). Regulatory roles of these newly identified phosphorylations in these processes need to be examined in further studies.…”

“…reesei (Rodriguez‐Iglesias & Schmoll, 2019). Protein kinases were also found to participate in regulating lignocellulase production (Lin et al, 2019; Wang et al, 2017).…”