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

Industrial lignocellulolytic 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 variety, making it a promising candidate for side stream utilization. In the present study, the cellular detoxification of furfural, 5-hydroxymethylfurfural (HMF), levulinic acid, ferulic acid and vanillin was studied at the transcriptome level. A. oryzae RIB40 was grown in the presence of different inhibitors commonly found in the lignocellulolytic side streams and RNA sequencing was utilized to investigate the different transcriptomic changes in response to the inhibitors. The main cause of the transcriptomic response in all conditions was the xenobiotic-induced formation of reactive oxygen species (ROS). Apart from levulinic acid, all conditions showed strong initial suppression of metabolic pathways relating to cell proliferation, ribosomal functions and 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 xenobiotics. 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 stronger and more severe stress response in A. oryzae RIB40. Additionally, we identified several promising target genes for future strain engineering. Overall design: Strain RIB40 was grown overnight at about 28°C with 220 rpm in shake flasks containing 250 mL Czapek-Dox with 2 % D-fructose. Mycelia was harvested on Miracloth and washed with Czapek-Dox. Equal portions of mycelia were transferred to flasks containing 50 mL Czapek-Dox with 2 % D-fructose and with or without a defined IC50 concentration of furfural, 5-hydroxymethylfurfural, vanillin, ferulic acid or levulinic acid. The cultures were incubated in rotary shakers for 24 h at 220 rpm. Total RNA of three biological replicates (quadruple for the control) was extracted at five different time points, 0 h, 1 h, 3 h, 5 h, and 24 h. The sample quality of 3 h and 5 h samples of ferulic acid and levulinic acid was not sufficient and those samples were excluded from the data set.