Deciphering Transcriptional Regulatory Mechanisms Associated with Hemicellulose Degradation in Neurospora crassa

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs).<jats:named-content content-type="genus-species">Neurospora crassa</jats:named-content>, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis to<jats:named-content content-type="genus-species">N. crassa</jats:named-content>grown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes in<jats:named-content content-type="genus-species">N. crassa</jats:named-content>, we identified a TF (XLR-1;<jats:italic>x</jats:italic>y<jats:italic>l</jats:italic>an degradation<jats:italic>r</jats:italic>egulator<jats:italic>1</jats:italic>) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 in<jats:named-content content-type="genus-species">Aspergillus</jats:named-content>and<jats:named-content content-type="genus-species">Trichoderma</jats:named-content>species, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion of<jats:italic>xlr-1</jats:italic>in<jats:named-content content-type="genus-species">N. crassa</jats:named-content>abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in<jats:named-content content-type="genus-species">N. crassa</jats:named-content>and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.</jats:p>

Bioethanol Production from Brewers Spent Grains Using a Fungal Consolidated Bioprocessing (CBP) Approach

“…In addition, the recognition of other lignocellulosic components such as hemicellulose might be crucial in triggering optimal secretion of cellulases through some form of signalling. This could be similar to the importance of the XLR-1 xylan degradation regulator (found in an alternative fungal species Neurospora crassa) with regard to its requirement of induction of other cellulolytic encoding genes [5]. Retaining the A. oryzae fungal species but substituting the original S. cerevisiae NCYC479 yeast strain partner with the NCYC2592 strain resulted in maximal ethanol concentrations of only ca.…”

“…CBP involves the conversion of lignocellulose into the required products in one step, without the addition of enzymes. Most attempts at CBP have utilised individual organisms, such as thermo-tolerant yeast strains [5] or bacteria (e.g. species of Clostridia; [6]).…”

Transcriptional Regulation and Responses in Filamentous Fungi Exposed to Lignocellulose

“…In the Δxlr-1 mutant, growth on cellulose and cellulolytic activity is only slightly affected [56]. Instead in N. crassa, two other Zn 2 Cys 6 family transcription factors CLR-1 and CLR-2 were found to be the predominant regulators of the expression of cellulaseencoding genes [45].…”

“…In N. crassa, cellobiose is the inducer primarily of cellulases [55]. Xylose in N. crassa induced fewer hemicellulase-encoding genes than a xylan polymer indicating that additional small molecules from hemicellulose are required for the full induction response or that the size or structure of the polymer is important [56]. Section II of this chapter will describe the response of fungi to xylan and other complex polymers (containing many different small molecule inducers).…”

“…That study defined the 'pectin regulon' and compared this to the 'Avicel regulon' from Coradetti et al [45] and the 'xylan regulon' -the xylan study was a replication of the microarray experiment from Sun et al [56] using RNAseq. All three substrates significantly induced a common set of 29 genes and three quarters of these genes encoded CAZymes.…”