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Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass.

Young J, Chung D, Bomble YJ, Himmel ME, Westpheling J - Biotechnol Biofuels (2014)

Bottom Line: A deletion of the celA gene resulted in a dramatic reduction in the microorganism's ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass.A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell.The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Georgia, Athens, Georgia ; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.

ABSTRACT

Background: Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms described to date, and have the ability to grow on lignocellulosic biomass without conventional pretreatment. Different species vary in their abilities to degrade cellulose, and the presence of CelA, a bifunctional glycoside hydrolase that contains a Family 48 and a Family 9 catalytic domain, correlates well with cellulolytic ability in members of this genus. For example, C. hydrothermalis, which does not contain a CelA homolog, or a GH48 Family or GH9 Family glycoside hydrolase, is the least cellulolytic of the Caldicellulosiruptor species so far described. C. bescii, which contains CelA and expresses it constitutively, is among the most cellulolytic. In fact, CelA is the most abundant extracellular protein produced in C. bescii. The enzyme contains two catalytic units, a Family 9A-CBM3c processive endoglucanase and a Family 48 exoglucanase, joined by two Family 3b carbohydrate-binding domains. Although there are two non-reducing end-specific Family 9 and three reducing end-specific Family 48 glycoside hydrolases (producing primarily glucose and cellobiose; and cellobiose and cellotriose, respectively) in C. bescii, CelA is the only protein that combines both enzymatic activities.

Results: A deletion of the celA gene resulted in a dramatic reduction in the microorganism's ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass. A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell. In contrast, it appears that no other enzymes in the C. bescii secretome can compensate for the loss of exoglucanase activity. The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains.

Conclusions: The exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.

No MeSH data available.


Cellulolytic activity (reducing sugars released byin vitroextracellular protein) of the extracellular fraction ofCaldicellulosiruptor besciigrown on cellobiose, on carboxymethylcellulose (CMC) (gray bars) after 1 hour and Avicel (white bars) after 24 hours incubation with the extracellular fraction of wild-type, JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains.
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Fig4: Cellulolytic activity (reducing sugars released byin vitroextracellular protein) of the extracellular fraction ofCaldicellulosiruptor besciigrown on cellobiose, on carboxymethylcellulose (CMC) (gray bars) after 1 hour and Avicel (white bars) after 24 hours incubation with the extracellular fraction of wild-type, JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains.

Mentions: CMC is a soluble form of cellulose specifically used for estimating endoglucanase activity. Avicel, a microcrystalline form of cellulose, is primarily used to estimate exoglucanase activity. Because CelA is a bifunctional enzyme with both endoglucanase and exoglucanase activity, both substrates were used to assay cellulolytic activity in the celA mutant. Extracellular proteins from the control cultures and the celA deletion strain grown on cellobiose were tested for cellulolytic activity. The activity of the wild-type and parent strain resulted in comparable sugar release, ranging from around 0.65 to 0.75 μg/ml sugar released from CMC, and 1.3 to 1.4 μg/ml sugar released on Avicel (Figure 4). The celA deletion strain showed a similar profile on CMC (0.6 μg/ml sugar released) to that of the wild-type and parent strains; however, a dramatic reduction in enzyme activity was observed on Avicel (approximately 0.1 μg/ml sugar released). The presence of endoglucanase activity in the mutant suggests that this activity, attributed to the GH9 family domain of CelA, is redundant in the genome and may partially compensate for the loss of CelA [27]. A recent study showed that the endoglucanase activity of the GH9 domain provides substrate for the exoglucanase activity of the GH48 domain [15], suggesting that this is the rate-limiting activity for this enzyme. The apparent need for an abundant amount of endoglucanase activity may explain the redundancy of these genes in the genome and the upregulation of their RNA transcripts during growth on biomass [19]. Interestingly, the Family 9 glycoside hydrolase of Clostridium phytofermentans was determined to be essential for growth on filter paper [27]. C. phytofermentans contains twice as many (108) glycosyl hydrolases as C. bescii, but contains only one GH9 family endoglucanase [27]. If the activity of the GH9 enzyme is rate-limiting, the redundancy of these enzymes in C. bescii may ensure enough GH9 non-reducing end-specific digestion of the substrate to allow reducing end-specific enzymes to deconstruct the biomass.Figure 4


Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass.

Young J, Chung D, Bomble YJ, Himmel ME, Westpheling J - Biotechnol Biofuels (2014)

Cellulolytic activity (reducing sugars released byin vitroextracellular protein) of the extracellular fraction ofCaldicellulosiruptor besciigrown on cellobiose, on carboxymethylcellulose (CMC) (gray bars) after 1 hour and Avicel (white bars) after 24 hours incubation with the extracellular fraction of wild-type, JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195899&req=5

Fig4: Cellulolytic activity (reducing sugars released byin vitroextracellular protein) of the extracellular fraction ofCaldicellulosiruptor besciigrown on cellobiose, on carboxymethylcellulose (CMC) (gray bars) after 1 hour and Avicel (white bars) after 24 hours incubation with the extracellular fraction of wild-type, JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains.
Mentions: CMC is a soluble form of cellulose specifically used for estimating endoglucanase activity. Avicel, a microcrystalline form of cellulose, is primarily used to estimate exoglucanase activity. Because CelA is a bifunctional enzyme with both endoglucanase and exoglucanase activity, both substrates were used to assay cellulolytic activity in the celA mutant. Extracellular proteins from the control cultures and the celA deletion strain grown on cellobiose were tested for cellulolytic activity. The activity of the wild-type and parent strain resulted in comparable sugar release, ranging from around 0.65 to 0.75 μg/ml sugar released from CMC, and 1.3 to 1.4 μg/ml sugar released on Avicel (Figure 4). The celA deletion strain showed a similar profile on CMC (0.6 μg/ml sugar released) to that of the wild-type and parent strains; however, a dramatic reduction in enzyme activity was observed on Avicel (approximately 0.1 μg/ml sugar released). The presence of endoglucanase activity in the mutant suggests that this activity, attributed to the GH9 family domain of CelA, is redundant in the genome and may partially compensate for the loss of CelA [27]. A recent study showed that the endoglucanase activity of the GH9 domain provides substrate for the exoglucanase activity of the GH48 domain [15], suggesting that this is the rate-limiting activity for this enzyme. The apparent need for an abundant amount of endoglucanase activity may explain the redundancy of these genes in the genome and the upregulation of their RNA transcripts during growth on biomass [19]. Interestingly, the Family 9 glycoside hydrolase of Clostridium phytofermentans was determined to be essential for growth on filter paper [27]. C. phytofermentans contains twice as many (108) glycosyl hydrolases as C. bescii, but contains only one GH9 family endoglucanase [27]. If the activity of the GH9 enzyme is rate-limiting, the redundancy of these enzymes in C. bescii may ensure enough GH9 non-reducing end-specific digestion of the substrate to allow reducing end-specific enzymes to deconstruct the biomass.Figure 4

Bottom Line: A deletion of the celA gene resulted in a dramatic reduction in the microorganism's ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass.A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell.The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Georgia, Athens, Georgia ; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.

ABSTRACT

Background: Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms described to date, and have the ability to grow on lignocellulosic biomass without conventional pretreatment. Different species vary in their abilities to degrade cellulose, and the presence of CelA, a bifunctional glycoside hydrolase that contains a Family 48 and a Family 9 catalytic domain, correlates well with cellulolytic ability in members of this genus. For example, C. hydrothermalis, which does not contain a CelA homolog, or a GH48 Family or GH9 Family glycoside hydrolase, is the least cellulolytic of the Caldicellulosiruptor species so far described. C. bescii, which contains CelA and expresses it constitutively, is among the most cellulolytic. In fact, CelA is the most abundant extracellular protein produced in C. bescii. The enzyme contains two catalytic units, a Family 9A-CBM3c processive endoglucanase and a Family 48 exoglucanase, joined by two Family 3b carbohydrate-binding domains. Although there are two non-reducing end-specific Family 9 and three reducing end-specific Family 48 glycoside hydrolases (producing primarily glucose and cellobiose; and cellobiose and cellotriose, respectively) in C. bescii, CelA is the only protein that combines both enzymatic activities.

Results: A deletion of the celA gene resulted in a dramatic reduction in the microorganism's ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass. A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell. In contrast, it appears that no other enzymes in the C. bescii secretome can compensate for the loss of exoglucanase activity. The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains.

Conclusions: The exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.

No MeSH data available.