Transcriptome analysis of Aspergillus oryzae RIB40 under chemical stress reveals mechanisms of adaptation to fungistatic compounds of lignocellulosic side streams

Abstract

Background: Industrial lignocellulosic side streams are considered an attractive carbon source for the cultivation of biotechnologically important fungi, although the presence of toxic pretreatment by-products is a major challenge yet to be overcome. Aspergillus oryzae is a filamentous fungus with a large secretion capacity, high tolerance for toxins, and a wide substrate range, making it a promising candidate for side stream utilization. In the present study, the cellular mechanisms of tolerance against furfural, 5-hydroxymethylfurfural (HMF), levulinic acid, ferulic acid, and vanillin were studied at the transcriptome level.

Results: A. oryzae RIB40 was grown in the presence of different inhibitors commonly found in lignocellulosic side streams, and RNA sequencing was utilized to investigate the transcriptomic changes in response to the inhibitors. Analysis of the transcriptomic response in all conditions indicates that a large fraction of differentially expressed genes responded to the inhibitor-induced formation of reactive oxygen species (ROS). Apart from levulinic acid, all inhibitors showed strong initial suppression of metabolic pathways related to cell cycle, ribosome functions, protein folding, and sorting in the endoplasmic reticulum. Genes associated with cellular detoxification, namely, NAD(P)H-dependent oxidoreductases and efflux transporters, such as the ATP-Binding Cassette (ABC) transporters and major facilitator superfamily (MFS) transporters, showed strong upregulation upon exposure to the inhibitors.

Conclusions: The results obtained provide important insights into the stress response of A. oryzae to the xenobiotic compounds and their cellular detoxification. Aldehydic inhibitors, especially HMF, caused a strong and severe stress response in A. oryzae RIB40. Additionally, we identified several highly upregulated uncharacterized genes upon exposure to the inhibitors. These genes serve as promising targets for strain engineering to build robust industrial strains capable of utilizing lignocellulosic side streams as feedstock.

Keywords: Detoxification; Filamentous fungi; Furaldehydes; Industrial side stream; Lignocellulosic inhibitors; Oxidoreductases; Phenolics; Transcriptome; Transporter proteins; Weak acid.

Fig. 1

Determination of IC₅₀ values for the selected inhibitors. Growth profiles of Aspergillus oryzae RIB40 on solid media supplemented with different inhibitors found in lignocellulosic hydrolysates. Cultures were grown on CZA medium supplemented with 2% D-fructose and increasing concentrations of the inhibitory compounds. Plates were incubated at + 28 °C for seven days. Control plates without supplementation of the inhibitors were made in quadruplicate. Three biological replicates were prepared for each concentration of the inhibitor. The white number in the upper right corner of each plate indicates the inhibitor concentration (mM). FF, furfural; Van, vanillin; FA, ferulic acid; LA, levulinic acid; HMF, hydroxymethylfurfural; GA, gallic acid; CA, cinnamic acid; SA, salicylic acid; SyA, syringic acid

Fig. 2

Venn diagrams of differentially expressed genes of each inhibitor one hour after induction (a) upregulated genes (b) downregulated genes

Fig. 3

Heatmaps of LFC values of the most upregulated oxidoreductases one hour after induction under levulinic acid (LA), ferulic acid (FA), vanillin, furfural, and HMF in comparison to the control strain at different time points. LFC values in the heatmaps are capped from − 5 to 10

Fig. 4

Sequence similarity network with an E-value cut-off of 10^–60 for reported fungal amino acid sequences of oxidoreductases (EC: 1.-.-.-). The sequences were colored based on characterized activity; the shape distinguishes the A. oryzae sequences (diamond) from sequences of characterized S. cerevisiae (circle) and other fungal proteins (square). Bolded sequences represent genes primarily involved in chemical stress responses, with some included to provide functional context within the clusters. Sc, Saccharomyces cerevisiae; Pc, Phanerochaete chrysosporium; Mr, Monascus ruber

Fig. 5

Heatmaps of LFC of the most highly upregulated A. oryzae ABC transporter genes under levulinic acid (LA), ferulic acid (FA), vanillin, furfural, and HMF in comparison to the control strain at different time points. LFC values in the heatmaps are capped from − 4 to 8

Fig. 6

Overview of the genes proposed for ferulic acid and vanillin (a) metabolic pathways in A. oryzae (b) LFC values of the genes. Black arrows indicate a known homolog from A. niger where the corresponding genes have been characterized. White arrows indicate a suggested conversion where the corresponding genes are unknown. Multiple arrows indicate multiple-step conversions. Black line indicates ring cleavage. LFC values in the heatmaps are capped from -5 to 10. Genes after the gap belong to the CoA-dependent β-oxidative pathway