Systems approaches to predict the functions of glycoside hydrolases during the life cycle of Aspergillus niger using developmental mutants ∆brlA and ∆flbA

Abstract

Background: The 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.

Results: In 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.

Conclusion: The 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.

Fig 1. Morphology of N402 (A), ∆flbA (B) and ∆brlA (C) during growth on agar plates.

The wild-type strain N402 forms aerial hyphae and conidiophores with black conidia, clearly visible from the top view (top) and schematic representation (bottom). The mutant strain ∆flbA forms only aerial hyphae that collapse in the colony center. Mutant strain ∆brlA lacks sporulation and forms conidial stalks that extend into the air, as clearly visible in the side view (middle).

Fig 2. Growth of strains N402 (squares), ∆flbA (triangles) and ∆brlA (dots) in batch cultures.

The biomass (A) in dry weight and protein content of the culture filtrate (B) (Bradford assay), given as mean ± SE of measurements performed for two biological duplicates.

Fig 3. Enzyme activities detected in culture filtrate of batch cultures of strains N402 (squares), ∆flbA (triangles) and ∆brlA (dots).

Protease activity (A), total hydrolytic activity releasing galactose (B) and glucose (C) from the isolated A. niger cell walls and GH exo-activity releasing N-acetyl-glucosamine from GlcNAc-β-pNP (D). Values are given as mean ± SE of measurements performed for two biological duplicates.

Fig 4. Carbohydrate composition of the cell walls of strains N402 (A), ∆flbA (B) and ∆brlA (C).

Values are given as mean ± SE in % of total moles of carbohydrate as detected in isolated cell walls of two independent growth experiments.