Identification and manipulation of Neurospora crassa genes involved in sensitivity to furfural

Abstract

Background Biofuels derived from lignocellulosic biomass are a viable alternative to fossil fuels required for transportation. Following plant biomass pretreatment, the furan derivative furfural is present at concentrations which are inhibitory to yeasts. Detoxification of furfural is thus important for efficient fermentation. Here, we searched for new genetic attributes in the fungus Neurospora crassa that may be linked to furfural tolerance. The fact that furfural is involved in the natural process of sexual spore germination of N. crassa and that this fungus is highly amenable to genetic manipulations makes it a rational candidate for this study. Results Both hypothesis-based and unbiased (random promotor mutagenesis) approaches were performed to identify N. crassa genes associated with the response to furfural. Changes in the transcriptional profile following exposure to furfural revealed that the affected processes were, overall, similar to those observed in Saccharomyces cerevisiae . N. crassa was more tolerant (by ~ 30%) to furfural when carboxymethyl cellulose was the main carbon source as opposed to sucrose, indicative of a link between carbohydrate metabolism and furfural tolerance. We also observed increased tolerance in a Δ cre - 1 mutant (CRE-1 is a key transcription factor that regulates the ability of fungi to utilize non-preferred carbon sources). In addition, analysis of aldehyde dehydrogenase mutants showed that ahd - 2 (NCU00378) was involved in tolerance to furfural as well as the predicted membrane transporter NCU05580 ( flr - 1 ), a homolog of FLR1 in S. cerevisiae . Further to the rational screening, an unbiased approach revealed additional genes whose inactivation conferred increased tolerance to furfural: (i) NCU02488, which affected the abundance of the non-anchored cell wall protein NCW-1 (NCU05137), and (ii) the zinc finger protein NCU01407. Conclusions We identified attributes in N. crassa associated with tolerance or degradation of furfural, using complementary research approaches. The manipulation of the genes involved in furan sensitivity can provide a means for improving the production of biofuel producing strains. Similar research approaches can be utilized in N. crassa and other filamentous fungi to identify additional attributes relevant to other furans or toxic chemicals.

Lineage-specific genes are clustered with allorecognition loci and respond to G × E factors regulating the switch from asexual to sexual reproduction inNeurospora

“…Expression of 22 orphan genes required that at least one type of carbon resource was present in the media, while expression of 56 other orphan genes was only detected in the absence of carbon. Revisiting expression data collected from experiments investigating N. crassa ’s tolerance to furfural (Feldman et al 2019) identified that 245, 239, 257, and 232 orphan genes exhibited measurable levels of expression in the wild-type condition, DMSO (the carbon blank control), furfural, and HMF treatments ( Tables S7, S8 ). Within the 291 genes expressed in either furfural or HMF cultures or both, 61 were not expressed in the wild-type condition.…”

“…Accordingly, many more het-like genes (46 vs. 15) were significantly up-regulated (P < 0.05) on MSM than on BM during that first branching stage. (Wang et al 2014;Wang, Miguel-Rojas, et al 2019) on media with various carbon resources (Wang et al 2015;Feldman et al 2019). 342 orphan genes were expressed at measurable levels in at least two stages in each of the three developmental processes (Tables S6-S7), including eight stages of sexual development on SCM (salmon pink, totaling 484 orphan genes with measurable expression), four stages of asexual growth on BM (beige, totaling 457 orphan genes with measurable expression) and four stages of asexua-sexual growth on MSM (light blue, totaling 489 orphan genes with measurable expression).…”

“…Orphan genes are generally not functionally annotated, mainly due to the lack of homologous references in well studied genomes. To determine the functional roles of Neurospora orphan genes ,we revisited recent high quality transcriptomics studies on N. crassa, covering almost all morphological stages in the N. crassa life cycle, cultures under different conditions and carbon or nitrogen resources as well as knockout mutants of key regulatory genes (Coradetti et al 2012;Znameroski et al 2012;Coradetti et al 2013;Lehr et al 2014;Wang et al 2014;Wu et al 2014;Craig et al 2015;Wang et al 2015;Xiong et al 2017;Feldman et al 2019;Sun et al 2019;Wang, Miguel-Rojas, et al 2019). We observed a significant enrichment of clustering of orphan and het-like genes and, within some of those clusters, highly coordinated regulation in response to arbon resources, light, and temperature conditions.…”

“…To assess environmental impacts on expression of orphan genes, recent available data on 37C bird from this lab (GSE168995) and a 240-minute time course of asexual growth in response to darkness and light stimulation (Wu et al 2014) were revisited. To assess possible roles of orphan genes in metabolic regulation, transcriptomics data from mycelia exposed to 5 different carbon resources from crop residues (Wang et al 2015) and from mycelia in response to non-preferred carbon sources such as furfural and 5-hydroxymethyl furfural (HMF; (Feldman et al 2019) were also examined separately. To assess mutation in transcription factors, including adv-1, pp-1, and ada-6, previous transcriptomics data (Fischer et al 2018;Sun et al 2019) were also analyzed with LOX v1.6 (Zhang et al 2010).…”

Pyrolyzed Substrates Induce Aromatic Compound Metabolism in the Post-fire Fungus, Pyronema domesticum

“…Pyronema may simply exist as dormant ascospores or sclerotia that require the heat and/or chemical changes associated with fire to break dormancy and initiate germination ( Emerson, 1947 ; El-Abyad and Webster, 1968a ). For example, plant-derived polysaccharides such as hemicellulose burned at high temperatures result in the formation of furfural, which triggers germination of N. crassa ascospores ( Eilers and Sussman, 1970 ; Feldman et al, 2019 ). One recent hypothesis suggests that pyrophilous fungi may live as endophytes for the majority of their life history, abandoning their plant hosts after they are killed by fire ( Raudabaugh et al, 2020 ).…”