Systems Approaches to Predict the Functions of Glycoside Hydrolases during the Life Cycle of Aspergillus niger Using Developmental Mutants ∆brlA and ∆flbA

Abstract

BackgroundThe filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis.ResultsIn this study, we used developmental mutants (ΔflbA and ΔbrlA) which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced.ConclusionThe combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall modification, which contributes to understanding cell wall remodeling mechanisms during development.

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger

“…anafp transcription levels are also depicted for batch cultivations in bioreactors with ΔflbA and ΔbrlA mutants, respectively [33]. Samples have been taken at four time points: t1–t4, exponential growth phase, 16 h, 60 h, 140 h post carbon depletion.…”

“…Samples have been taken at four time points: t1–t4, exponential growth phase, 16 h, 60 h, 140 h post carbon depletion. In C) mRNA levels at different zones of a plated culture are depicted [33, 34]. Whereas zone 1 (z1) marks the center of the colony consisting, i.e.…”

“…The aconidial, slow growing phenotype of Δ flbA is characterized by a fluffy appearance of the colony due to excessive formation of aerial hyphae. It lacks conidiophores, has thinner cell walls compared to the wild type, shows signs of cell wall stress and collapses due to autolytic disintegration of aerial hyphae in the center of the colony [33, 34]. As shown in Fig 2C, xylose-grown cultures of N402 displayed highest anafp expression in the center of the colony (zone 1, about 13.7×10 3 AU), which gradually decreased towards the colony periphery (zone 3, about 9.5×10 3 AU and zone 5, about 3.5×10 3 AU).…”

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

“…The up-regulation of glycoside hydrolases (GHs) and proteases has been demonstrated in transcriptomic and proteomic analysis of A. niger and A. nidulans grown under carbon starvation [32,[44][45][46]. Like GT exposure, carbon starvation is a type of stress that triggers a response by filamentous fungi.…”

“…Of the glycoside hydrolases up-regulated in response to GT, 22 out of 30 were identified in the extracellular proteome of A. niger grown on carbon limitation. These include GlaA, AamA, AgdA, CrhD, BgtB, GelD, BxgA, AglA, NagA, An03g00500, MsdS, EglB, AgdB, AglB, An14g04240 (no SigP), AmyC, MndA, An07g07700 (no SigP), GelA, DfgC (no SigP), An08g07350, and CtcA [46]. Due to the presence of a N-terminal signal peptide (SigP) in the majority (83%) of GHs up-regulated after GT addition, we hypothesise that this response is a consequence of a carbon limitation or a glucose demand induced by GT, whereby A. niger responds by synthesizing these enzymes in order to breakdown complex carbohydrates.…”