The roles of the zinc finger transcription factors XlnR, ClrA and ClrB in the breakdown of lignocellulose by Aspergillus niger

Genes encoding the key transcription factors (TF) XlnR, ClrA and ClrB were deleted from Aspergillus niger and the resulting strains were assessed for growth on glucose and wheat straw, transcription of genes encoding glycosyl hydrolases and saccharification activity. Growth of all mutant strains, based in straw on measurement of pH and assay of glucosamine, was impaired in relation to the wild type (WT) strain although deletion of clrA had less effect than deletion of xlnR or clrB. Release of sugars from wheat straw was also lowered when culture filtrates from TF deletion strains were compared with WT culture filtrates. Transcript levels of cbhA, eglC and xynA were measured in all strains in glucose and wheat straw media in batch culture with and without pH control. Transcript levels from cbhA and eglC were lowered in all mutant strains compared to WT although the impact of deleting clrA was not pronounced with expression of eglC and had no effect on xynA. The impact on transcription was not related to changes in pH. In addition to impaired growth on wheat straw, the ?xlnR strain was sensitive to oxidative stress and displayed cell wall defects in the glucose condition suggesting additional roles for XlnR. The characterisation of TFs, such as ClrB, provides new areas of improvement for industrial processes for production of second generation biofuels

Transcriptomic responses of mixed cultures of ascomycete fungi to lignocellulose using dual RNA-seq reveal inter-species antagonism and limited beneficial effects on CAZyme expression

Gaining new knowledge through fungal monoculture responses to lignocellulose is a widely used approach that can lead to better cocktails for lignocellulose saccharification (the enzymatic release of sugars which are subsequently used to make biofuels). However, responses in lignocellulose mixed cultures are rarely studied in the same detail even though in nature fungi often degrade lignocellulose as mixed communities. Using a dual RNA-seq approach, we describe the first study of the transcriptional responses of wild-type strains of Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum in two and three mixed species shake-flask cultures with wheat straw. Based on quantification of species-specific rRNA, a set of conditions was identified where mixed cultures could be sampled so as to obtain sufficient RNA-seq reads for analysis from each species. The number of differentially-expressed genes varied from a couple of thousand to fewer than one hundred. The proportion of carbohydrate active enzyme (CAZy) encoding transcripts was lower in the majority of the mixed cultures compared to the respective straw monocultures. A small subset of P. chrysogenum CAZy genes showed five to ten-fold significantly increased transcript abundance in a two-species mixed culture with T. reesei. However, a substantial number of T. reesei CAZy transcripts showed reduced abundance in mixed cultures. The highly induced genes in mixed cultures indicated that fungal antagonism was a major part of the mixed cultures. In line with this, secondary metabolite producing gene clusters showed increased transcript abundance in mixed cultures and also mixed cultures with T. reesei led to a decrease in the mycelial biomass of A. niger. Significantly higher monomeric sugar release from straw was only measured using a minority of the mixed culture filtrates and there was no overall improvement. This study demonstrates fungal interaction with changes in transcripts, enzyme activities and biomass in the mixed cultures and whilst there were minor beneficial effects for CAZy transcripts and activities, the competitive interaction between T. reesei and the other fungi was the most prominent feature of this study

Development of an unmarked gene deletion system for the filamentous fungi [i]Aspergillus niger[/i] and [i]Talaromyces versatilis[/i]

In this article, we present a method to delete genes in filamentous fungi that allows recycling of the selection marker and is efficient in a nonhomologous end-joining (NHEJ)-proficient strain. We exemplify the approach by deletion of the gene encoding the transcriptional regulator XlnR in the fungus Aspergillus niger. To show the efficiency and advantages of the method, we deleted 8 other genes and constructed a double mutant in this species. Moreover, we showed that the same principle also functions in a different genus of filamentous fungus (Talaromyces versatilis, basionym Penicillium funiculosum). This technique will increase the versatility of the toolboxes for genome manipulation of model and industrially relevant fungi

Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing

A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall–degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA) to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions

The role of carbon starvation in the induction of enzymes that degrade plant-derived carbohydrates in Aspergillus niger

AbstractFungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6h of exposure to wheat straw was very different from the response at 24h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24h of exposure to wheat straw, were also induced after 6h exposure. Importantly, over a third of the genes induced after 6h of exposure to wheat straw were also induced during 6h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes

Induction model based on the sequential expression of responsive genes.

<p>The sequence of events is illustrated and key events are numbered. The upper panel represents the transcriptional events in <i>A. niger</i> upon exposure (0–6 h) to straw represented by filled ovals. Lack of easily-available carbon source leads to the alleviation of CreA repression (represented by the arrow above CreA) and induction of a subset of starvation-induced genes represented by <i>cbhB</i>. At 6–9 h exposure to straw (middle panel), the expressed hydrolases and other enzymes (examples named in the Figure.) act upon the wheat straw, releasing small quantities of inducing sugars such as xylose (filled pentamer) as well as glucose (filled hexamer). Transporters for the sugars are induced (indicated by the trans-membrane cylinders and un-filled large arrow). By 9 hours (lower panel) the presence of xylose has caused activation of XlnR and, thereby, large scale expression of hydrolases genes. Also induced by 9 hours, in an XlnR-independent manner, is the hydrophobic binding protein HsbA. The hydrophobin HfbD is induced by 12 hours. A physical association of hydrophobic binding proteins with straw and degradative enzymes is hypothesised and represented. Note that the functionality of XlnR and CreA is indicated by attachment to recognition sequences in target promoters and is meant only to indicate the functional control of those promoters by the transcription factors. Modifications to those transcription factors (e.g. phosphorylation of XlnR in <i>A. oryzae </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002875#pgen.1002875-Noguchi1" target="_blank">[65]</a>) may occur without necessarily implying that their location changes.</p

Straw-induced non-CAZy genes.

<p>Non-CAZy genes strongly induced (≥20 x) and expressed (≥50 RPKM) after 24 h in Straw. An03g06560 and An08g09880 have not been annotated in the ATCC 1015 genome but were found on chromosome 6_1 (5′-1494949 - 1403261-3′) and 8_2 (5′-2365138 - 2364987 - 3′) respectively. Annotation are from CADRE <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002875#pgen.1002875-MabeyGilsenan1" target="_blank">[66]</a> (<a href="http://www.cadre-genomes.org.uk/index.html" target="_blank">http://www.cadre-genomes.org.uk/index.html</a>). RPKM values are from the combined mapping of three biologically-independent samples under each condition, values for each individual sample are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002875#pgen.1002875.s009" target="_blank">Table S2</a>. All genes listed showed a statistically significant induction when switched from glucose to straw (p>0.001).</p

Straw-induced CAZy genes.

<p>CAZy genes strongly induced (≥20 x) and expressed (≥50 RPKM) after 24 h in Straw. An02g02540 has not been annotated in the ATCC 1015 genome but was found on chromosome 4_2 (5′-669026 - 667767-3′). Annotation are from CADRE <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002875#pgen.1002875-MabeyGilsenan1" target="_blank">[66]</a> (<a href="http://www.cadre-genomes.org.uk/index.html" target="_blank">http://www.cadre-genomes.org.uk/index.html</a>). RPKM values are from the combined mapping of three biologically-independent samples under each condition, values for each individual sample are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002875#pgen.1002875.s009" target="_blank">Table S2</a>. All genes listed showed a statistically significant induction when switched from glucose to straw (p>0.001).</p

Sense and antisense transcription from TID_53176.

<p>(A) Alignment of RNA-seq reads to the TID_53176 genome region under each condition. Reads represented in blue are antisense, those in red are sense. The Figure was constructed using the Integrative Genomics Viewer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002875#pgen.1002875-Robinson1" target="_blank">[62]</a>. (B) Oligo(dT) primed RT-PCR using TID_53176 specific primers. The expected band size from the spliced sense transcript is 411 bp and the size of the non-spliced antisense transcript is 524 bp. The red line under the gene model in panel A indicates the amplified region. (C) Strand-specific RT-PCR. One of the standard PCR primers, with an added sequence tag (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002875#pgen.1002875.s012" target="_blank">Table S5</a>), was used to synthesise cDNA from one strand only and then the PCR step was performed by using the tagged sequencing primer together with the opposing gene-specific primer. The larger band is only seen in the antisense-specific reaction, confirming it does represent an antisense transcript. The smaller band is the only band present in the sense-specific reaction.</p