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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

Time course expression ofP. ostreatus aao1-6andaad1genes 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.
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Fig3: Time course expression ofP. ostreatus aao1-6andaad1genes 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.

Mentions: In order to verify whether the observed AAO and AAD protein accumulation is a consequence of alteration in the expression of the corresponding genes, we constructed specific primers to monitor their expression levels by quantitative real-time polymerase chain reaction (RT-PCR). RNA was extracted from P. ostreatus hyphae at different time points following exposure to HMF. Following the kinetics of expression, we observed a significant and substantial increase in all AAO and AAD mRNA levels (Figure 3), with three patterns of expression. The first group (aao1-3 and aad1) was rapidly transcribed following exposure to HMF and expression levels increased at least twofold relative to the control as early as 30 min after exposure to the furan. The second group (aao5-6) showed increased expression levels after 2 h of exposure to HMF. Both these groups, the maximum expression change ranged from 4- to 150-fold, which was observed following 8 or 24 h of exposure to HMF. After this time period, transcript abundance of both of these groups of genes declined. aao4 exhibited a third expression pattern characterized by a slower increase in expression, starting only 8 h after exposure to HMF. Although the observed increase in the rate of aao4 expression was slower than the other groups of genes, it exhibited the most dramatic change (approximately 300-fold) in its expression level, at 24 h after HMF exposure.Figure 3


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)

Time course expression ofP. ostreatus aao1-6andaad1genes 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Time course expression ofP. ostreatus aao1-6andaad1genes 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.
Mentions: In order to verify whether the observed AAO and AAD protein accumulation is a consequence of alteration in the expression of the corresponding genes, we constructed specific primers to monitor their expression levels by quantitative real-time polymerase chain reaction (RT-PCR). RNA was extracted from P. ostreatus hyphae at different time points following exposure to HMF. Following the kinetics of expression, we observed a significant and substantial increase in all AAO and AAD mRNA levels (Figure 3), with three patterns of expression. The first group (aao1-3 and aad1) was rapidly transcribed following exposure to HMF and expression levels increased at least twofold relative to the control as early as 30 min after exposure to the furan. The second group (aao5-6) showed increased expression levels after 2 h of exposure to HMF. Both these groups, the maximum expression change ranged from 4- to 150-fold, which was observed following 8 or 24 h of exposure to HMF. After this time period, transcript abundance of both of these groups of genes declined. aao4 exhibited a third expression pattern characterized by a slower increase in expression, starting only 8 h after exposure to HMF. Although the observed increase in the rate of aao4 expression was slower than the other groups of genes, it exhibited the most dramatic change (approximately 300-fold) in its expression level, at 24 h after HMF exposure.Figure 3

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