UC Berkeley

Lessons in Plant Cell Wall Degradation by the Ascomycete Model Fungus, Neurospora crassa

by

Vincent W. Wu

Doctor of Philosophy in Microbiology

University of California, Berkeley

Professor N. Louise Glass, Chair

In order to mitigate the negative environmental effects of anthropogenic global climate change, renewable resources must be developed to replace fossil fuels. One potential fossil fuel replacement platform utilizes plant biomass from agricultural waste as a feedstock to generate fuels and high value products. Plant cell wall polysaccharides including cellulose, hemicellulose and pectin are broken down into fermentable sugars that can be converted to compounds of interest by microorganisms. Filamentous fungi are capable of both depolymerizing polysaccharides and converting fermentable sugars to high value products. However, further engineering is required to generate strains of filamentous fungi that can efficiently secrete both polysaccharide-degrading enzymes and/or synthesized compounds of interest. This engineering is only possible through a better understanding of the metabolic biology of these organisms. This dissertation describes the mechanisms by which the model filamentous fungus Neurospora crassa regulates and produces the enzymes required for depolymerization of plant cell wall polysaccharides.

We investigated pyroglutamate, a unique post-translational modification, and have determined the mechanism of catalysis, as well as its role in the secretion of plant cell wall degrading enzymes. Pyroglutamate modification is catalyzed by two glutaminyl cyclases localized in the endoplasmic reticulum. Secreted proteins with either an N-terminal glutamine or glutamate are likely to have this terminal amino acid converted to a pyroglutamate. Pyroglutamate provides proteins with protection from native N. crassa proteases. The modification also provides structural stability for enzymes within glycosyl hydrolase family 7, including CBH-1 and other important cellulases. The amino acid sequences of glutaminyl cyclases are highly conserved within the fungal kingdom, including critical amino acids required for catalysis and substrate binding. These data indicate that our findings regarding the role of pyroglutamate in N. crassa may be broadly applicable to other fungal genera.

Transcriptional profiling of N. crassa exposed to various components of the plant cell wall resulted in the identification of a number of candidate transcription factors and sugar transporters involved plant cell wall deconstruction. Results from these experiments also suggest that N. crassa is responsive to a diverse set of signaling sugars. These sugars induce transcription of plant polysaccharide lytic enzymes. The responses to these sugars can explain transcriptional responses of N. crassa to parent plant cell wall polymers from which sugars are derived.

Analysis of candidate transcription factors revealed a complete set of regulators responsible for positively regulating expression of plant cell wall degrading enzymes in the N. crassa genome. Regulation of hemicellulytic and pectinolytic enzymes is governed by PDR-1, PDR-2, and ARA-1 along with the previously described xylanase regulator XLR-1. These transcription factors are conserved homologs of RhaR, GaaR and Ara-1, respectively, from Aspergillus and other species. We found additional transcription factors that we named HEM-1 and MAN-1 that play a minor role in the positive regulation of hemicellulases. Transcriptional data from engineered strains lacking these transcription factors, along with data from ∆clr-1, ∆clr-2 and ∆xlr-1, indicate that these sugar responsive regulators have overlapping regulons. Surprisingly, none of these transcription factors affect the expression of one another.

DNA affinity purification sequencing (DAPseq) was used to assay direct DNA-binding of a number of N. crassa transcription factors. This assay is comparable to chromatin immune-precipitation sequencing (ChIPseq). Direct binding data of XLR-1 and CLR-2 from both DAPseq and ChIPseq indicate that the same core set of hemicellulases and cellulases are directly regulated by the two transcription factors, respectively. Additionally, DAPseq binding sites for nitrogen and sulfur metabolism regulators NIT-2, CYS-3 and NUC-1 confirm previously described targets for these transcription factors.

Direct binding data from DAPseq provides new insights into the mechanisms of function of the carbon catabolite repressor CRE-1. Contrary to previous research, DAPseq data shows that CRE-1 represses the expression of cellulases through the repression of cellobiose transporters cdt-1, cdt-2, and cbt-1. Repression of hemicellulases and pectinases expression may occur in a similar manner.

DAPseq data also revealed the mechanism of VIB-1 influence in plant cell wall degradation. VIB-1 not only directly regulates expression of genes involved in vegetative incompatibility, phosphatase production and protease production described in previous literature, but VIB-1 also directly binds the promoters of clr-2, xlr-2, pdr-2, ara-1 and others key regulators of polysaccharide deconstruction. Direct binding of VIB-1 to the clr-2 promoter is consistent with previously published data. VIB-1 directly regulates many genes involved in nutrient acquisition of all types and may be a key regulator that integrates signals from many types of nutrient limitation.

The complementary CRE-1 and VIB-1 data provide a new model for induction of plant cell wall degrading enzymes in ascomycete fungi. This model suggests that VIB-1 may be responsible for sensing of extracellular plant cell wall polysaccharides by promoting basal transcription of polysaccharide degrading enzymes capable of releasing signaling sugars that increase production of select enzymes. The regulation of transporters by CRE-1 occurs upstream of this VIB-1 induction, allowing the two transcription factors to work cooperatively.