Expression of glycoside hydrolases in Aspergillus niger

Enzymes from filamentous fungi have a key role in degradation of the most abundant biopolymers found in nature, cellulose and hemicelluloses. For this reason, these enzymes are of great interest in the industrial conversion of lignocellulosic substrates into biofuels. The production of plant cell wall degrading enzymes is regulated mainly at the transcriptional level in filamentous fungi but little is known about the signalling pathways and transcription factors (TFs) involved in this regulation in Aspergillus niger. RNA-sequencing analysis has been previously carried out to investigate the transcriptional changes that occur when A. niger is transferred from the simple carbon source glucose onto the complex lignocellulosic biomass wheat straw. This has highlighted the up-regulation in transcript level of genes encoding some glycosyl hydrolase (GH) enzymes as well as hydrophobic surface interacting proteins (HSIPs) that may be involved in the interface between lignocellulosic biomass and A. niger. Genes encoding the key TFs XlnR, ClrA and ClrB were deleted from A. 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, bglB, 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, bglB 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 ΔxlnR strain was sensitive to oxidative stress and displayed cell wall defects in the glucose condition suggesting additional roles for XlnR. Phosphorylation is a key reversible modification that regulates protein function, subcellular localization, complex formation, activation of TFs and cell signalling pathways. A phosphoproteomic study was carried out on both the WT and the ΔxlnR deletion strains of A. niger in order to identify key regulators of the signalling pathways involved in the breakdown of a lignocellulosic substrate, wheat straw. The analysis consisted of comparing the phosphoproteome profiles of the strains when grown in glucose with the phosphoproteome profile of the same strains when exposed to wheat straw for 6h, 12h and 24h. The results suggested a difference in the phosphoproteome profiles of the two strains when exposed to both glucose and wheat straw. These data may provide new information on the importance of XlnR in the regulation of expression of GHs but also in controlling the environment to which A. niger is exposed depending on the nutrient availability. To investigate the role of HSIPs in the induction of A. niger response to wheat straw, single gene deletion strains for hfbD, hyp1 and hsbA as well as the double deletion strain for hfbD and hyp1 have been constructed. The expression of some genes encoding GH enzymes was then followed in these strains using qRT-PCR. The results showed that the transcript levels of the GH genes studied were lowered in the HSIPs deletion strains when compared to the wild-type strain, when the cultures were transferred from glucose medium to wheat straw. These results suggest that HSIPs may have a role in the utilisation of lignocellulosic biomass in A. niger. The precise nature of such a role as well as the characterisation of new TFs, such as ClrB, provides new areas of improvement for industrial processes for production of second generation biofuels

Transcriptional regulation and responses in filamentous fungi exposed to lignocellulose

Biofuels derived from lignocellulose are attractive alternative fuels but their production suffers from a costly and inefficient saccharification step that uses fungal enzymes. One route to improve this efficiency is to understand better the transcriptional regulation and responses of filamentous fungi to lignocellulose. Sensing and initial contact of the fungus with lignocellulose is an important aspect. Differences and similarities in the responses of fungi to different lignocellulosic substrates can partly be explained with existing understanding of several key regulators and their mode of action, as will be demonstrated for Trichoderma reesei, Neurospora crassa and Aspergillus spp. The regulation of genes encoding Carbohydrate Active enZymes (CAZymes) is influenced by the presence of carbohydrate monomers and short oligosaccharides, as well as the external stimuli of pH and light. We explore several important aspects of the response to lignocellulose that are not related to genes encoding CAZymes, namely the regulation of transporters, accessory proteins and stress responses. The regulation of gene expression is examined from the perspective of mixed cultures and models are presented for the nature of the transcriptional basis for any beneficial effects of such mixed cultures. Various applications in biofuel technology are based on manipulating transcriptional regulation and learning from fungal responses to lignocelluloses. Here we critically access the application of fungal transcriptional responses to industrial saccharification reactions. As part of this chapter, selected regulatory mechanisms are also explored in more detail

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

Novel combinations of agents targeting translation that synergistically inhibit fungal pathogens

A range of fungicides or antifungals are currently deployed to control fungi in agriculture or medicine, but resistance to current agents is growing so new approaches and molecular targets are urgently needed. Recently, different aminoglycoside antibiotics combined with particular transport inhibitors were found to produce strong, synergistic growth-inhibition of fungi, by synergistically increasing the error rate of mRNA translation. Here, focusing on translation fidelity as a novel target for combinatorial antifungal treatment, we tested the hypothesis that alternative combinations of agents known to affect the availability of functional amino acids would synergistically inhibit growth of major fungal pathogens. We screened 172 novel combinations against three phytopathogens (Rhizoctonia solani, Zymoseptoria tritici, Botrytis cinerea) and three human pathogens (Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus), showing that 48 combinations synergistically inhibited growth. Of these, 23 combinations were effective against more than one pathogen, including combinations comprising food-and-drug approved compounds, e.g., quinine with bicarbonate, and quinine with hygromycin. These combinations (fractional inhibitory combination [FIC] index [less than] 0.5) gave up to 100% reduction of fungal growth yield at concentrations of agents which, individually, had negligible effect. No synergy was evident against bacterial, plant or mammalian cells, indicating specificity for fungi. Mode-of-action analyses for quinine + hygromycin indicated that synergistic mistranslation was the antifungal mechanism. That mechanism was not universal as bicarbonate exacerbated quinine action by increasing drug uptake. The study unveils chemical combinations and a target process with potential for control of diverse fungal pathogens, and suggests repurposing possibilities for several current therapeutics

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism

Responses to hydric stress in the seed-borne necrotrophic fungus Alternaria brassicicola

Alternaria brassicicola is a necrotrophic fungus causing black spot disease and is an economically important seed-borne pathogen of cultivated brassicas. Seed transmission is a crucial component of its parasitic cycle as it promotes long-term survival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A. brassicicola pathosystem, showed that the level of susceptibility of the fungus to water stress strongly influenced its seed transmission ability. In this study, we gained further insights into the mechanisms involved in the seed infection process by analyzing the transcriptomic and metabolomic responses of germinated spores of A. brassicicola exposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteins that are assumed to be involved in cellular dehydration tolerance, was established in A. brassicicola based on the expression data and additional structural and biochemical criteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-like proteins showed that they were affected in their transmission to A. thaliana seeds, although their aggressiveness on host vegetative tissues remained intact

Expression of glycoside hydrolases in Aspergillus niger

Enzymes from filamentous fungi have a key role in degradation of the most abundant biopolymers found in nature, cellulose and hemicelluloses. For this reason, these enzymes are of great interest in the industrial conversion of lignocellulosic substrates into biofuels. The production of plant cell wall degrading enzymes is regulated mainly at the transcriptional level in filamentous fungi but little is known about the signalling pathways and transcription factors (TFs) involved in this regulation in Aspergillus niger. RNA-sequencing analysis has been previously carried out to investigate the transcriptional changes that occur when A. niger is transferred from the simple carbon source glucose onto the complex lignocellulosic biomass wheat straw. This has highlighted the up-regulation in transcript level of genes encoding some glycosyl hydrolase (GH) enzymes as well as hydrophobic surface interacting proteins (HSIPs) that may be involved in the interface between lignocellulosic biomass and A. niger. Genes encoding the key TFs XlnR, ClrA and ClrB were deleted from A. 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, bglB, 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, bglB 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 ΔxlnR strain was sensitive to oxidative stress and displayed cell wall defects in the glucose condition suggesting additional roles for XlnR. Phosphorylation is a key reversible modification that regulates protein function, subcellular localization, complex formation, activation of TFs and cell signalling pathways. A phosphoproteomic study was carried out on both the WT and the ΔxlnR deletion strains of A. niger in order to identify key regulators of the signalling pathways involved in the breakdown of a lignocellulosic substrate, wheat straw. The analysis consisted of comparing the phosphoproteome profiles of the strains when grown in glucose with the phosphoproteome profile of the same strains when exposed to wheat straw for 6h, 12h and 24h. The results suggested a difference in the phosphoproteome profiles of the two strains when exposed to both glucose and wheat straw. These data may provide new information on the importance of XlnR in the regulation of expression of GHs but also in controlling the environment to which A. niger is exposed depending on the nutrient availability. To investigate the role of HSIPs in the induction of A. niger response to wheat straw, single gene deletion strains for hfbD, hyp1 and hsbA as well as the double deletion strain for hfbD and hyp1 have been constructed. The expression of some genes encoding GH enzymes was then followed in these strains using qRT-PCR. The results showed that the transcript levels of the GH genes studied were lowered in the HSIPs deletion strains when compared to the wild-type strain, when the cultures were transferred from glucose medium to wheat straw. These results suggest that HSIPs may have a role in the utilisation of lignocellulosic biomass in A. niger. The precise nature of such a role as well as the characterisation of new TFs, such as ClrB, provides new areas of improvement for industrial processes for production of second generation biofuels

Combining analytical approaches for better lignocellulosic biomass degradation: a way of improving fungal enzymatic cocktails?

International audienc

High-throughput strain identification and production of fungal enzymatic cocktails for the valorisation of lignocellulosic biomass

International audienc

High-throughput strain identification and production of fungal enzymatic cocktails for the valorisation of lignocellulosic biomass

International audienc