UC Berkeley

A substantive transition from an unsustainable fossil material and energy economy to a robust and sustainable biofuels and biomaterials economy will require a fundamental change in the economics of cellulosic biomass deconstruction to sugars with secreted fungal enzymes. To supply these enzymes on the scale required by global biorefinery industry will likely necessitate rational engineering of fungal production strains to produce a wider diversity of enzymes at higher yield and on more diverse substrates. This will, in turn require more complete knowledge of how expression of fungal enzymes, particularly cellulases, are regulated. To further elucidate general features of fungal enzyme regulation and identify new regulatory proteins affecting enzyme expression, I carried out a research program on transcriptional regulation of cellulases in the model filamentous fungus Neurospora crassa with comparative experiments in the evolutionarily divergent species Aspergillus nidulans.

A screen of predicted transcription factor mutants from the N. crassa deletion collection identified several genes with previously unknown effects on cellulase expression on cellulose. When N. crassa is transferred to inducing substrates such as cellulose, approximately 200 genes are transcriptionally induced, including several dozen genes that encode known carbohydrate active enzymes (CAZy genes) with expression increased as much as several thousand fold over non-inducing starvation conditions. This transcriptional induction was compromised in several transcription factor deletion mutants. Two deletion mutants, for the genes that I named named cellulose degradation regulator 1 and cellulose degradation regulator 2 (CLR-1 and CLR-2), and entirely lost their transcriptional response to cellulose.

Functional CLR-1 was required for efficient cellobiose utilization and cellulase secretion. Though CLR-1 transcript abundance increases on cellulose, constitutive expression of CLR-1 at or above levels observed in inducing conditions was insufficient to stimulate cellulase secretion in non-inducing conditions, suggesting a requirement for post-translational activation of CLR-1. Functional CLR-2 is required for cellulose utilization but not cellobiose utilization. CLR-2 expression also increases dramatically on cellulose and constitutive expression of CLR-2 at comparably high levels was sufficient for cellulase secretion in non- inducing conditions. However, not all β-glucosidases and cellobiose transporters were expressed in CLR-2 constitutive expression strains. CLR-2 expression was CLR-1 dependent. I proposed a regulatory model in which a cellobiose sensing activates CLR-1, which in turn induces gene expression of CLR-2, the primary direct regulator of cellulase expression.

Analysis of these transcription factors in A. nidulans revealed conserved and divergent aspects, with a drastically reduced role for the CLR-1 ortholog ClrA, which regulates only a single major cellulase. The CLR-2 ortholog ClrB was not dependent on ClrA and was not only required for cellulose utilization but for cellobiose sensing and utilization as well. Constitutive expression of ClrB was not sufficient for constitutive cellulase secretion. Cellulases were strongly induced when WT A. nidulans was exposed to cellobiose, but not when it was exposed to mannobiose. When ClrB constitutive/over-expression strains were exposed to mannobiose, major cellulases were strongly induced.

Global transcriptional comparisons of WT and mutant strains of N. crassa and A. nidulans also provided new insights into shared and divergent strategies for cellulose degradation, particularly by the lytic polysaccharide monooxgenase (LPMO) enzyme class. Differing expression profiles between LPMO subclasses between N. crassa and A. nidulans may be reflective of still unappreciated differences in enzymatic mechanism or preferred substrate between LPMO classes.

To address emerging difficulties in correlating sequence similarity and functional conservation for fungal transcription factors, both in the above studies and contemporary studies on other regulators of plant cell wall degrading enzymes, I initiated a search for additional components of the cellobiose sensing system. I constructed an antibiotic resistance construct under regulation of CLR-1 and CLR-2, then selected for mutants with antibiotic resistance in carefully selected non- inducing, non-repressing conditions, isolating several mutants of interest. Bulk segregant analysis on the first of these mutants identified a new repressor of CLR-1 activation (NCU05846). Further characterization of NCU05846 and additional components of the cellobiose-sensing network may further enhance our ability to manipulate cellulase gene expression in N. crassa. That knowledge may also enrich our understanding of fungal nutrient sensing, and enable more nuanced and predictive analysis of how such systems are conserved across fungal species in general and highly productive industrial strains in particular.