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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

Profiles of secreted and cellularP. ostreatusproteins 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.
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Fig2: Profiles of secreted and cellularP. ostreatusproteins 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.

Mentions: To investigate the enzymatic processes involved in HMF transformation by P. ostreatus, a time course experiment in which changes in the profiles of secreted and intracellular proteins as a result of HMF exposure was monitored. Proteins were concentrated from the extracellular fraction at different stages of HMF transformation (8, 24, and 48 h after HMF addition to the media, corresponding to approximately 5%, 50%, and 100% transformation, respectively). Surprisingly, a significant increase in the abundance of an approximately 75-kDa secreted protein was evident from samples at the beginning of the transformation process (only 8 h after HMF addition). Its abundance peaked at a time corresponding to 50% HMF transformation and diminished once all the HMF was detoxified (Figure 2). The approximately 75-kDa band was sequenced and identified as having a high coverage and score of 6 aryl-alcohol oxidase proteins (AAO; EC 1.1.3.7): 69649, 82653, 93955, 114510, 116309, and 121882, designated aao1-6, respectively. AAOs are secreted peroxide-producing flavoenzymes and members of the glucose-methanol-choline oxidase (GMC) oxidoreductase superfamily [29-31]. P. ostreatus has a large AAO, a gene family consisting of at least 36 genes [32]. Phylogenetic analysis of the AAOs in P. ostreatous revealed that they cluster into two groups (Additional file 3).Figure 2


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)

Profiles of secreted and cellularP. ostreatusproteins 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Profiles of secreted and cellularP. ostreatusproteins 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.
Mentions: To investigate the enzymatic processes involved in HMF transformation by P. ostreatus, a time course experiment in which changes in the profiles of secreted and intracellular proteins as a result of HMF exposure was monitored. Proteins were concentrated from the extracellular fraction at different stages of HMF transformation (8, 24, and 48 h after HMF addition to the media, corresponding to approximately 5%, 50%, and 100% transformation, respectively). Surprisingly, a significant increase in the abundance of an approximately 75-kDa secreted protein was evident from samples at the beginning of the transformation process (only 8 h after HMF addition). Its abundance peaked at a time corresponding to 50% HMF transformation and diminished once all the HMF was detoxified (Figure 2). The approximately 75-kDa band was sequenced and identified as having a high coverage and score of 6 aryl-alcohol oxidase proteins (AAO; EC 1.1.3.7): 69649, 82653, 93955, 114510, 116309, and 121882, designated aao1-6, respectively. AAOs are secreted peroxide-producing flavoenzymes and members of the glucose-methanol-choline oxidase (GMC) oxidoreductase superfamily [29-31]. P. ostreatus has a large AAO, a gene family consisting of at least 36 genes [32]. Phylogenetic analysis of the AAOs in P. ostreatous revealed that they cluster into two groups (Additional file 3).Figure 2

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