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 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.纤维素分解真菌（Cellulolytic fungi）已演化出一套复杂的调控网络，以维系生长及水解酶合成所需营养物质的精准平衡。当真菌暴露于纤维素环境时，纤维素酶基因的转录水平会先快速升高，随后下降。然而，这种钟形表达模式背后的分子机制仍不明晰。本研究以丝状真菌（filamentous fungus）粗糙脉孢霉（Neurospora crassa）的蛋白激酶缺失突变体库进行系统筛选，旨在寻找纤维素酶基因表达模式异常的突变株。我们发现，敲除stk-12（NCU07378）会导致纤维素酶产量显著提升，且主要纤维素酶基因的转录本高丰度表达时期显著延长。上述结果表明，stk-12作为负调控因子发挥关键作用，通过下调纤维素酶基因的转录以抑制水解酶的过量表达，避免能量浪费。转录组分析（Transcriptional profiling）显示，Δstk-12突变体中纤维素酶基因的高表达状态可维持56小时，而野生型（Wild-type, WT）菌株仅能维持8小时。在纤维素培养基上生长3天后，Δstk-12突变体的纤维素酶基因转录本水平是野生型的3.3倍；其中，编码转录激活因子的clr-2在Δstk-12突变体中表达上调，而编码两个纤维素酶阻遏蛋白的res-1与rca-1则表达下调。最终，Δstk-12突变体的总纤维素酶产量较野生型提升7倍。上述结果有力表明，敲除stk-12会导致纤维素酶表达调控系统的功能失调。进一步分析显示，STK-12可直接靶向IGO-1以调控纤维素酶的合成。TORC1通路（TORC1 pathway）至少可通过抑制STK-12的功能来促进纤维素酶的产生；同时，STK-12与CRE-1可通过平行通路共同抑制纤维素酶基因的转录。本研究阐明了纤维素诱导过程中纤维素酶基因在转录水平上的阻遏机制，并为工业真菌菌株的改良提供了全新策略。