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

Feldman D, Kowbel DJ, Glass NL, Yarden O, Hadar Y - Biotechnol Biofuels (2015)

Bottom Line: In this study, we analyzed the ability of P. ostreatus to tolerate and metabolize HMF and investigated relevant molecular pathways associated with these processes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, The R.H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, POB 12, Rehovot, 76100 Israel.

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.

No MeSH data available.


Related in: MedlinePlus

In vitrogeneration of peroxide is increased in the extracellular fraction ofP. ostreatusafter 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.
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Fig5: In vitrogeneration of peroxide is increased in the extracellular fraction ofP. ostreatusafter 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.

Mentions: Secreted AAOs oxidize alcohols and aldehydes while generating H2O2. We therefore also monitored peroxide production as another indication for AAO activity [21,36]. Indeed, an increase in H2O2 concentration was observed in the samples containing veratryl alcohol as a substrate, 24 h after HMF addition to the fungal culture. Furthermore, peroxide levels in the reaction mixture were maintained at approximately 700 nM/μl, even 4 days after exposure. By contrast, at the same time points, H2O2 was not detected in control cultures (Figure 5A). These results are in agreement with the changes in AAO activity levels described above (Figure 4). When HMF was introduced as a substrate to the assay mixture, peroxide levels increased to approximately 70 nM/μl as soon as 8 h after exposure (Figure 5B). The maximum level of H2O2 detected (approximately 200 nM/μl) occurred 2 to 4 days after HMF addition to the fungal culture. These results could be explained by changes in preferential expression of the different AAOs (Figure 3), suggesting that AAOs whose expression is induced shortly after exposure to HMF may have a higher affinity for HMF than for veratryl alcohol.Figure 5


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

Feldman D, Kowbel DJ, Glass NL, Yarden O, Hadar Y - Biotechnol Biofuels (2015)

In vitrogeneration of peroxide is increased in the extracellular fraction ofP. ostreatusafter 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403834&req=5

Fig5: In vitrogeneration of peroxide is increased in the extracellular fraction ofP. ostreatusafter 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.
Mentions: Secreted AAOs oxidize alcohols and aldehydes while generating H2O2. We therefore also monitored peroxide production as another indication for AAO activity [21,36]. Indeed, an increase in H2O2 concentration was observed in the samples containing veratryl alcohol as a substrate, 24 h after HMF addition to the fungal culture. Furthermore, peroxide levels in the reaction mixture were maintained at approximately 700 nM/μl, even 4 days after exposure. By contrast, at the same time points, H2O2 was not detected in control cultures (Figure 5A). These results are in agreement with the changes in AAO activity levels described above (Figure 4). When HMF was introduced as a substrate to the assay mixture, peroxide levels increased to approximately 70 nM/μl as soon as 8 h after exposure (Figure 5B). The maximum level of H2O2 detected (approximately 200 nM/μl) occurred 2 to 4 days after HMF addition to the fungal culture. These results could be explained by changes in preferential expression of the different AAOs (Figure 3), suggesting that AAOs whose expression is induced shortly after exposure to HMF may have a higher affinity for HMF than for veratryl alcohol.Figure 5

Bottom Line: In this study, we analyzed the ability of P. ostreatus to tolerate and metabolize HMF and investigated relevant molecular pathways associated with these processes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, The R.H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, POB 12, Rehovot, 76100 Israel.

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.

No MeSH data available.


Related in: MedlinePlus