Ethanol bio-fuel derived from lignocellulosic biomass is a viable alternative to fossil-fuel-based transportation fuels. Pretreatment with a thermo and/or chemical process is necessary to make such sources available for enzymatic hydrolysis/fermentation but is accompanied by the production of compounds such as furaldehydes and phenolics that can inhibit efficient biomass utilization by fermentation. Profiling of the Neurospora crassa transcriptional response to furfural or 5-hydroxymethyl-2-furaldehyde (HMF) revealed changes in the expression of 2190 genes, determining that multiple processes, including oxidative stress, ribosomal biogenesis, amino acid synthesis, transport and RNA metabolism were affected. In a complementary approach, we also screened for strains that confer altered tolerance or furfural detoxification capabilities. Based on the proven functions of relevant genes from Sacchromyces cerevisiae, Escherichia coli and Cupriavidus basilensis, 25 N. crassa deletion mutants were assessed for growth in the presence of furfural (EC50). A strain in which a gene encoding for the membrane transporter NCU05580 (designated flr-1) was deleted, conferred a ~30% increase in sensitivity to furfural (but not to other furans such as HMF and furfuryl alcohol). The mutant exhibited slow growth and was defective in aerial hyphae production. Overexpression of flr-1 under a strong constitutive promoter (cpc-1Δ2uorf) fully complemented the abnormal phenotype of ΔNCU05580. The overexpression strain grew 60% faster than the wild type in the presence of 20mM furfural (EC80), confirming the functional link between flr-1 and furfural sensitivity. In addition, we have isolated several furan-tolerant mutants following random insertion of the cpc-1Δ2uorf promoter cassette. Taken together, manipulation of genes involved in furan sensitivity/detoxification can provide a means for improving strains used for lignocellulose fermentation.
