Detoxification of 5-hydroxymethylfurfural by the Pleurotus ostreatus lignolytic enzymes aryl alcohol oxidase and dehydrogenase

Abstract

Background: Current large-scale pretreatment processes for lignocellulosic biomass are generally accompanied by the formation of toxic degradation products, such as 5-hydroxymethylfurfural (HMF), which inhibit cellulolytic enzymes and fermentation by ethanol-producing yeast. Overcoming these toxic effects is a key technical barrier in the biochemical conversion of plant biomass to biofuels. Pleurotus ostreatus, a white-rot fungus, can efficiently degrade lignocellulose. In this study, we analyzed the ability of P. ostreatus to tolerate and metabolize HMF and investigated relevant molecular pathways associated with these processes.

Results: P. ostreatus was capable to metabolize and detoxify HMF 30 mM within 48 h, converting it into 2,5-bis-hydroxymethylfuran (HMF alcohol) and 2,5-furandicarboxylic acid (FDCA), which subsequently allowed the normal yeast growth in amended media. We show that two enzymes groups, which belong to the ligninolytic system, aryl-alcohol oxidases and a dehydrogenase, are involved in this process. HMF induced the transcription and production of these enzymes and was accompanied by an increase in activity levels. We also demonstrate that following the induction of these enzymes, HMF could be metabolized in vitro.

Conclusions: Aryl-alcohol oxidase and dehydrogenase gene family members are part of the transcriptional and subsequent translational response to HMF exposure in P. ostreatus and are involved in HMF transformation. Based on our data, we propose that these enzymatic capacities of P. ostreatus either be integrated in biomass pretreatment or the genes encoding these enzymes may function to detoxify HMF via heterologous expression in fermentation organisms, such as Saccharomyces cerevisiae.

Keywords: 5-hydroxymethylfurfural (HMF); Aryl-alcohol dehydrogenase; Aryl-alcohol oxidase; Pleurotus ostreatus.

Figure 1

Relative growth of P. ostreatus in the presence of different concentrations of HMF. Different concentrations of HMF were added to GP solid media and linear growth of P. ostreatus was measured relative to a control lacking HMF. Bars indicate standard errors.

Figure 2

Profiles of secreted and cellular P. ostreatus proteins obtained from cultures grown in the presence of HMF. Secreted (A) and cellular (B) proteins were extracted from P. ostreatus 8, 24, and 48 h after addition of 30 mM of HMF to the media. The proteins were resolved by SDS-PAGE 4% to 12%. C: control cultures without HMF, H: cultures exposed to HMF. Arrows point to major visible difference in the profiles.

Figure 3

Time course expression of P. ostreatus aao1-6 and aad1 genes following the addition of HMF. The expression levels of aao1-6, aad1, and vp1 were monitored by real-time RT-PCR (for primers information see Additional file 4). RNA was extracted from P. ostreatus at different time points (0.5, 2, 8, 14, and 48 h) after addition of 30 mM HMF to the media. The expression levels calculated relative to β-tubulin, as the endogenous control, and represent the expression relative to control without HMF addition. Bars indicate standard errors.

Figure 4

In vitro AAO activity is increased in the extracellular fraction of P. ostreatus after addition of HMF to the medium. The activity of AAO following 1 mM veratryl alcohol addition as a substrate was monitored in free cell extracts of P. ostreatus, at different time points after addition of 30 or 20 mM HMF to the media. Bars indicate standard errors.

Figure 5

In vitro generation of peroxide is increased in the extracellular fraction of P. ostreatus after addition of HMF to the medium. Concentration of H₂O₂ generated during activity in vitro with 1 mM veratryl alcohol (A) or 10 mM HMF (B) over time in free cell extracts of P. ostreatus. The measurements were performed at different time points after addition of 30 mM HMF to the medium. Bars indicate standard errors.

Figure 6

Specific activity coupled with NAD(P)H in free cell extracts of P. ostreatus after addition of HMF to the medium. Depletion of NADPH (A) or NADH (B) was monitored over time in vitro with cell extracts of P. ostreatus with 10 mM HMF as a substrate. The measurements were performed at different time points after addition of 30 mM HMF to the culture.

Figure 7

Scheme for the enzymatic degradation of HMF by P. ostreatus. HMF is metabolized extracellularly by AAO to FDCA, which generates H₂O₂. These reactions probably involve an intermediate conversion of HMF to HMF acid and further conversion to unknown products. When HMF enters the cell, it is reduced by AAD with NADPH as a co-factor and metabolized to HMF alcohol. HMF alcohol can be secreted and accumulates extracellularly.