Current large scale pretreatment processes for lignocellulosic biomass are accompanied by the formation of degradation products, such as 5-hydroxymethylfurfural (HMF). HMF inhibits the cellulolytic enzymes and the ethanol-producing yeast. Overcoming these toxic effects is a key technical barrier in the biochemical conversion of biomass feedstock to biofuels. Pleurotus ostreatus, a white rot fungus, has a variety of secreted proteins involved in lignin biodegradation that include phenol oxidases and peroxidases, as well extracellular H2O2-producing enzymes such as aryl-alcohol oxidases (AAO) and intracellular aryl alcohol dehydrogenase (AAD).
P. ostreatuswas capable of metabolizing and detoxifying 30 mM of HMF within 48 hours to 2,5-bis-hydroxymethylfuran and 2,5-Furandicarboxylic acid, which subsequently allowed for normal yeast growth. The addition of HMF significantly elevated expression levels of AAOs (aao1-6) and AAD (aad1). Expression levels increased 30-300 fold by 24 hrs, and as fast as 30 min after exposure to HMF. Subsequently, the abundance of AAOs and AAD was increased in the presence of HMF. HMF metabolism was also followed in vitro with fungal extract pre-exposed to the compound. AADs activity was coupled with NADPH, and AAOs metabolized HMF while generating H2O2.
We conclude that AAO and AAD-encoding gene family members are part of the transcriptional and translational reaction to HMF. Functional redundancy exists between the genes and their products, even though their roles in the reaction may differ. We propose that P. ostreatus can serve as gene pool for heterologous expression in yeast, or be integrated in biomass pretreatment.
