RNAseq reveals hydrophobins that are involved in the adaptation of Aspergillus nidulans to lignocellulose

Abstract

Background: Sugarcane is one of the world's most profitable crops. Waste steam-exploded sugarcane bagasse (SEB) is a cheap, abundant, and renewable lignocellulosic feedstock for the next-generation biofuels. In nature, fungi seldom exist as planktonic cells, similar to those found in the nutrient-rich environment created within an industrial fermenter. Instead, fungi predominantly form biofilms that allow them to thrive in hostile environments.

Results: In turn, we adopted an RNA-sequencing approach to interrogate how the model fungus, Aspergillus nidulans, adapts to SEB, revealing the induction of carbon starvation responses and the lignocellulolytic machinery, in addition to morphological adaptations. Genetic analyses showed the importance of hydrophobins for growth on SEB. The major hydrophobin, RodA, was retained within the fungal biofilm on SEB fibres. The StuA transcription factor that regulates fungal morphology was up-regulated during growth on SEB and controlled hydrophobin gene induction. The absence of the RodA or DewC hydrophobins reduced biofilm formation. The loss of a RodA or a functional StuA reduced the retention of the hydrolytic enzymes within the vicinity of the fungus. Hence, hydrophobins promote biofilm formation on SEB, and may enhance lignocellulose utilisation via promoting a compact substrate-enzyme-fungus structure.

Conclusion: This novel study highlights the importance of hydrophobins to the formation of biofilms and the efficient deconstruction of lignocellulose.

Keywords: Biofilm; Fungi; Hydrolytic enzymes; Hydrophobin; Sugarcane bagasse.

Fig. 1

Growth of Aspergillus nidulans on steam-exploded sugarcane bagasse (SEB). a A. nidulans was grown in 1 % fructose liquid media for 24 h and then transferred to a semi-solid SEB media for 6–120 h at 37 °C. b Growth profile of A. nidulans grown on fructose (0 h) and post transfer to SEB for 24, 72, and 120 h shows the reduction of fungal growth and increased secretion post transfer to SEB. Presented are the mean total protein measurements of the solid and liquid fractions (plus one standard deviation) representative of fungal biomass and fungal secretion. c RNA-sequencing identifies the genes significantly up, or down, regulated post transfer to SEB for 6 or 12 h. d Venn analysis reveals a significant correlation in the modulation of transcription post 6 or 12 h growth on SEB

Fig. 2

Transcriptional analyses reveal how Aspergillus nidulans adapts to growth on SEB. a Significant transcriptional modulation of genes encoding for transcription factors involved in alternative carbon usage, starvation responses, and morphological adaptations post transfer to SEB. b Transcriptional induction of an array of CAZymes post transfer to SEB, in particular those from the GH families which target hemicellulose GH2, GH3, GH10, GH11, GH43, GH62) or lignin (GH61 now reclassified as AA9). c Transcriptional induction of numerous putative and characterised sugar transporter encoding genes post transfer to SEB. d RT-qPCR analysis of 17 putative sugar transporter encoding genes validates RNA-seq data. A heatmap of the RT-qPCR analysis showing the expression of 17 genes during growth on 1 % fructose, 0.1 % xylose, 1 % xylose, and 0.5 % SEB. The majority of genes showed higher expression levels at low xylose concentrations implying that they encoded putative high affinity transporters

Fig. 3

Involvement of hydrophobins in the growth of A. nidulans on SEB. a Transcriptional modulation of hydrophobin encoding genes post transfer to SEB reveals induction of rodA and dewC. b Radial growth on complete media is not affected by the absence of RodA or DewC. c Growth of A. nidulans on SEB is reduced in the absence of RodA or DewC. d Western blot showing the retention of RodA::mRFP within the solid fraction of the submerged cultivation of A. nidulans on SEB for 1–5 days. Arrows present the two RodA isoforms, potentially including a hydrophobin dimer. C denotes the coomassie stained 4–12 % Bis–Tris gel. Statistical significance: *p < 0.05

Fig. 4

Scanning electron microscopy (SEM) of A. nidulans biofilms grown on SEB. a SEB (0.5 %) was deposited on 12 mm adhesive discs and transferred into a 24 well plate containing liquid media (without any carbon source) plus 5 × 10⁵ conidia/ml for 48 h at 37 °C. Biofilms were washed, fixed, and sputter-coated with gold prior to SEM. b Wild-type A. nidulans forms a biofilm on the SEB particles (500×). c Absence of RodA or DewC results in the reduction in biofilm formation on SEB (1000×) and the alteration of the appearance of the hyphal surface (12,000×)

Fig. 5

Solid-state fermentation (SSF) of A. nidulans on SEB. a Representative image of SSF. Autoclaved and dried SEB was mixed with 5 ml of liquid media without any carbon source plus 1 × 10⁷ conidia and then incubated at 37 °C for 10 days. Proteins were extracted from solid SEB cultures. The resulting supernatants were used for the respective Megazyme assays. b Accumulative production of cellulolytic (blue) and xylanolytic (red) enzymes by the wild-type A. nidulans strain throughout the 10 days SSF. c Recovery of lignocellulolytic enzymes (predominantly xylanases) from the A. nidulans biofilm was increased in the absence of RodA or DewC, or the in the presence of the non-functional StuA1 mutation, during the SSF of SEB. Enzyme activity is presented in relation to the biomass of the fungal colony. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Redundancy in hydrophobin transcription during SSF of SEB. The absence of RodA, and in particular DewC, induced the transcription of alternative hydrophobin encoding genes (rodA and dewA-E). The regulation of multiple hydrophobin encoding genes was influenced by the presence of the non-functional StuA1 mutation. Presented is the relative expression of the hydrophobin encoding genes (cDNA hydrophobin/cDNA tubC) ± one standard deviation. Statistical significance: **p < 0.01, ***p < 0.001