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A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E.

Clarkson SM, Hamilton-Brehm SD, Giannone RJ, Engle NL, Tschaplinski TJ, Hettich RL, Elkins JG - Biotechnol Biofuels (2014)

Bottom Line: Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ.T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural.In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.

View Article: PubMed Central - PubMed

Affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA.

ABSTRACT

Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.

Results: Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.

Conclusions: T. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.

No MeSH data available.


Related in: MedlinePlus

24-h substrate and end product analysis.T. pseudethanolicus was grown at 65°C with 40 mM glucose and increasing concentrations of (A) furfural or (B) 5-HMF for 24 h. Culture supernatants were analyzed for glucose and end product formation by HPLC. Glucose consumption was determined by comparison with an uninoculated control. Error bars are the standard deviation of three replicate cultures.
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Fig3: 24-h substrate and end product analysis.T. pseudethanolicus was grown at 65°C with 40 mM glucose and increasing concentrations of (A) furfural or (B) 5-HMF for 24 h. Culture supernatants were analyzed for glucose and end product formation by HPLC. Glucose consumption was determined by comparison with an uninoculated control. Error bars are the standard deviation of three replicate cultures.

Mentions: The addition of furan aldehydes affected end-product formation by T. pseudethanolicus 39E, as determined by HPLC analysis (Figure 3). At 10 mM concentrations, more acetate and lactate are produced, while ethanol production remains constant. At furan aldehyde concentrations above 10 mM where growth is observed (15, 20, 30 mM furfural and 15, 20 mM 5-HMF), ethanol decrease and acetate increase are directly proportional, suggesting that acetyl coenzyme A is converted to acetate through phosphotransacetylase (PTA) and acetate kinase (AK) rather than serving as an electron acceptor for ethanol production via NAD(P)H-dependent bifunctional alcohol dehydrogenase (ADH) activity. T. pseudethanolicus 39E possesses seven ADHs, but ethanol is primarily produced from NADPH-dependent AdhB [34-36]. The oxidative branch of the pentose phosphate pathway is also present in 39E, which could supply NADPH [34]. Reduction of furfural/5-HMF to their corresponding alcohols during growth competes with ethanol production for electrons delivered by NADPH. This is also suggested by stoichiometric shifts in end products, where added aldehydes resulted in about a 0.5 times decrease in corresponding molar ethanol concentrations (that is, the 30 mM furfural addition resulted in a decrease of 15 mM ethanol versus the control). This shift in ethanol/acetate concentrations is consistent with an electron balance of one NAD(P)H per furan aldehyde and two NAD(P)H per ethanol. The reason for increased lactate with added furan aldehyde, especially 5-HMF, is less clear. Further redox imbalances from the presence of furfural or 5-HMF may direct more NADH generated from glycolysis to be oxidized via lactate dehydrogenase (LdhA). Furfural addition to a growing culture of the related thermophilic bacterium C. thermocellum also resulted in increased lactate production and cessation of ethanol production [37], although the reason for these changes is unknown. The mesophilic ethanologen S. cerevisiae has also been shown to remove furan aldehydes by reduction to their respective alcohols at the expense of ethanol production [38]. This has been shown to involve an upregulation of central carbon metabolism, especially the NADPH-generating pentose phosphate pathway [39], and downregulation of enzymes involved in ethanol formation, thereby increasing the availability of reducing equivalents for aldehyde detoxification [40].Figure 3


A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E.

Clarkson SM, Hamilton-Brehm SD, Giannone RJ, Engle NL, Tschaplinski TJ, Hettich RL, Elkins JG - Biotechnol Biofuels (2014)

24-h substrate and end product analysis.T. pseudethanolicus was grown at 65°C with 40 mM glucose and increasing concentrations of (A) furfural or (B) 5-HMF for 24 h. Culture supernatants were analyzed for glucose and end product formation by HPLC. Glucose consumption was determined by comparison with an uninoculated control. Error bars are the standard deviation of three replicate cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265447&req=5

Fig3: 24-h substrate and end product analysis.T. pseudethanolicus was grown at 65°C with 40 mM glucose and increasing concentrations of (A) furfural or (B) 5-HMF for 24 h. Culture supernatants were analyzed for glucose and end product formation by HPLC. Glucose consumption was determined by comparison with an uninoculated control. Error bars are the standard deviation of three replicate cultures.
Mentions: The addition of furan aldehydes affected end-product formation by T. pseudethanolicus 39E, as determined by HPLC analysis (Figure 3). At 10 mM concentrations, more acetate and lactate are produced, while ethanol production remains constant. At furan aldehyde concentrations above 10 mM where growth is observed (15, 20, 30 mM furfural and 15, 20 mM 5-HMF), ethanol decrease and acetate increase are directly proportional, suggesting that acetyl coenzyme A is converted to acetate through phosphotransacetylase (PTA) and acetate kinase (AK) rather than serving as an electron acceptor for ethanol production via NAD(P)H-dependent bifunctional alcohol dehydrogenase (ADH) activity. T. pseudethanolicus 39E possesses seven ADHs, but ethanol is primarily produced from NADPH-dependent AdhB [34-36]. The oxidative branch of the pentose phosphate pathway is also present in 39E, which could supply NADPH [34]. Reduction of furfural/5-HMF to their corresponding alcohols during growth competes with ethanol production for electrons delivered by NADPH. This is also suggested by stoichiometric shifts in end products, where added aldehydes resulted in about a 0.5 times decrease in corresponding molar ethanol concentrations (that is, the 30 mM furfural addition resulted in a decrease of 15 mM ethanol versus the control). This shift in ethanol/acetate concentrations is consistent with an electron balance of one NAD(P)H per furan aldehyde and two NAD(P)H per ethanol. The reason for increased lactate with added furan aldehyde, especially 5-HMF, is less clear. Further redox imbalances from the presence of furfural or 5-HMF may direct more NADH generated from glycolysis to be oxidized via lactate dehydrogenase (LdhA). Furfural addition to a growing culture of the related thermophilic bacterium C. thermocellum also resulted in increased lactate production and cessation of ethanol production [37], although the reason for these changes is unknown. The mesophilic ethanologen S. cerevisiae has also been shown to remove furan aldehydes by reduction to their respective alcohols at the expense of ethanol production [38]. This has been shown to involve an upregulation of central carbon metabolism, especially the NADPH-generating pentose phosphate pathway [39], and downregulation of enzymes involved in ethanol formation, thereby increasing the availability of reducing equivalents for aldehyde detoxification [40].Figure 3

Bottom Line: Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ.T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural.In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.

View Article: PubMed Central - PubMed

Affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA.

ABSTRACT

Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.

Results: Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.

Conclusions: T. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.

No MeSH data available.


Related in: MedlinePlus