CLR‐4, a novel conserved transcription factor for cellulase gene expression in ascomycete fungi

Abstract

Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and is an important part of the natural carbon cycle.Although induction of cellulases has been described for some saprobic filamentous fungi, the regulation of cellulase transcription is complex and many aspects are still poorly understood. Here, we identified and characterized the novel cellulase regulation factor NcCLR-4 in Neurospora crassa and its ortholog MtCLR-4 in Myceliophthora thermophila. Deletion of CLR-4 resulted in similarly defective cellulolytic enzyme production and activities. Transcriptome analyses of ΔNcclr-4/ΔMtclr-4 revealed the downregulation of genes encoding (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1) and key genes in the cAMP signaling pathway such as adenylate cyclase Nccr-1. Intracellular cAMP levels were markedly lower in ΔNcclr-4/ΔMtclr-4 than in wildtype during cellulose utilization. In electrophoretic mobility shift (EMSA) and DNase I footprinting assays, NcCLR-4/MtCLR-4 can directly bound to the promoters of Nccr-1/Mtcr-1 (encoding adenylyl cyclase). EMSAs also demonstrated that NcCLR-4/ MtCLR-4 could directly bound to clr-1 (encoding a key cellulase regulator), Mtclr-2 and Mtxyr-1 (encoding biomass deconstruction regulators). These findings about the novel cellulase expression regulators NcCLR-4 and MtCLR-4 enrich our understanding of how cellulose degradation is regulated and provide new targets for engineering fungi to deconstruct plant biomass in biorefineries.

Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose

“…Similarly, Hxk1 of S. cerevisiae was highly transcribed in cellobiose-grown cells [7]. Mycth_2297364 also showed elevated transcript levels in cells grown with glucose, starch, and Avicel compared with cells grown in starvation conditions [26,34]. These data indicate that Mycth_2297364 is the predominant isoenzyme when cells are grown with fermentable carbon sources.…”

“…2d). In previous studies, two genes encoding intracellular β-glucosidases were also induced by Avicel [26] and plant biomass [35], indicating that they are the most relevant enzymes for converting cellobiose to glucose when M. thermophila is cultured with either cellobiose or plant-derived biomass as the sole carbon source.…”

“…2c). Mycth_38704 (clr-2), Mycth_2305813 (clr-3), Mycth_2310145 (xlr-1), Mycth_114107 (cdt-2), and Mycth_84164 (clp1) are components of the ''Mt Avicel regulon" [26] and their orthologs in N. crassa are components of the "Nc Avicel regulon" [27]. CLR-2, CLR-3, XLR-1, CDT-2, and CLP1 are known to play essential roles in (hemi)-cellulose utilization by N. crassa [27][28][29][30][31].…”

Involvement of PaSNF1 in Fungal Development, Sterigmatocystin Biosynthesis, and Lignocellulosic Degradation in the Filamentous Fungus Podospora anserina

“…In addition, the transcription factors CLR-1 and CLR-2 were essential for growth on cellulose in N. crassa (Coradetti et al, 2012). Recent studies have shown that the CLR-4 acted as a positive activator of cellulase expression by directly binding to the promoters of CLR-1 and CLR-2 (Liu et al, 2019). CLR-3 repressed the cellulolytic response through CLR-1 (Huberman et al, 2017).…”

RCO-3 and COL-26 form an External-to-Internal Module that Regulates the Dual-Affinity Glucose Transport System in Neurospora Crassa

“…qRT-PCR was performed as previously described (94). The actin gene NCU04173 was used as an endogenous control for N. crassa, and Mycth_2314852 was used as an endogenous control for M. thermophila.…”