Analysis of a conserved cellulase transcriptional regulator reveals inducer‐independent production of cellulolytic enzymes in Neurospora crassa

Abstract

Cellulose is recalcitrant to deconstruction to glucose for use in fermentation strategies for biofuels and chemicals derived from lignocellulose. In Neurospora crassa, the transcriptional regulator, CLR-2, is required for cellulolytic gene expression and cellulose deconstruction. To assess conservation and divergence of cellulase gene regulation between fungi from different ecological niches, we compared clr-2 function with its ortholog (clrB) in the distantly related species, Aspergillus nidulans. Transcriptional profiles induced by exposure to crystalline cellulose were similar in both species. Approximately 50% of the cellulose-responsive genes showed strict dependence on functional clr-2/clrB, with a subset of 28 genes encoding plant biomass degrading enzymes that were conserved between N. crassa and A. nidulans. Importantly, misexpression of clr-2 under noninducing conditions was sufficient to drive cellulase gene expression, secretion, and activity in N. crassa, to a level comparable to wild type exposed to Avicel. However, misexpression of clrB in A. nidulans was not sufficient to drive cellulase gene expression under noninducing conditions, although an increase in cellulase activity was observed under crystalline cellulose conditions. Manipulation of clr-2 orthologs among filamentous fungi may enable regulated cellulosic enzyme production in a wide array of culture conditions and host strains, potentially reducing costs associated with enzyme production for plant cell wall deconstruction. However, this functionality may require additional engineering in some species.

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

“…Such regulators have been described, among others, for Aspergillus niger and Neurospora crassa, and could potentially activate a complete set of CAZymes simultaneously (10,78,79). Furthermore, potent heterologous enzymes, such as cellulases containing carbohydrate-binding domains (80), should be exploited to enhance the accessibility of the cellulose.…”

Transcriptional Regulation and Responses in Filamentous Fungi Exposed to Lignocellulose

“…Expression of clr-2 driven by a non-endogenous promoter) in N. crassa led to inducer independent increases in CAZyme-encoding gene expression, whereas similar expression of clrB in A. nidulans did not have such an effect [84]. Also, A. nidulans clrB cannot complement a clr-2 N. crassa strain [84]. In A. oryzae, the orthologue of CLR-2 called ManR was characterised as a regulator of genes encoding mannan degrading enzymes [85].…”

“…Orthologues of clr-1 and clr-2 have been identified in many fungal species [45] but some of the orthologues have been demonstrated to be functionally different. A. nidulans ClrB has more limited functions than CLR-2 in N. crassa [84]. Expression of clr-2 driven by a non-endogenous promoter) in N. crassa led to inducer independent increases in CAZyme-encoding gene expression, whereas similar expression of clrB in A. nidulans did not have such an effect [84].…”

“…A. nidulans ClrB has more limited functions than CLR-2 in N. crassa [84]. Expression of clr-2 driven by a non-endogenous promoter) in N. crassa led to inducer independent increases in CAZyme-encoding gene expression, whereas similar expression of clrB in A. nidulans did not have such an effect [84]. Also, A. nidulans clrB cannot complement a clr-2 N. crassa strain [84].…”

“…In A. oryzae, the orthologue of CLR-2 called ManR was characterised as a regulator of genes encoding mannan degrading enzymes [85]. Coradetti et al [84] suggested that the differences in cellulase gene regulation in filamentous fungi may reflect an ancient divergence in the regulatory mechanisms between the Sordariomycetes (includes N. crassa and T. reesei) and the Eurotiomycetes (includes Aspergillus spp.). The characterisation of the functions of the orthologues of clr-1 and clr-2 in T. reesei could substantiate whether the differences are related to an ancient evolutionary divergence or otherwise.…”