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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.


Related in: MedlinePlus

Growth of the wild-type, JWCB005 (ΔpyrFA), JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains on various carbon sources. (A, B) growth of the wild-type (closed circles), JWCB005 (open squares), JWCB018 (open circles), and JWCB029 (open triangles) strains on cellobiose. (C-F) Growth of the JWCB018 (solid bars), and JWCB029 (open bars) strains on (C)Populus trichocarpa (poplar), (D)Panicum virgatum L. (switchgrass), (E)Arabidopsis thaliana, and (F) Avicel. (G) Growth as a percentage reduction in cell numbers of the CelA deletion strain, JWCB029, compared to the background strain JWCB018.
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Fig2: Growth of the wild-type, JWCB005 (ΔpyrFA), JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains on various carbon sources. (A, B) growth of the wild-type (closed circles), JWCB005 (open squares), JWCB018 (open circles), and JWCB029 (open triangles) strains on cellobiose. (C-F) Growth of the JWCB018 (solid bars), and JWCB029 (open bars) strains on (C)Populus trichocarpa (poplar), (D)Panicum virgatum L. (switchgrass), (E)Arabidopsis thaliana, and (F) Avicel. (G) Growth as a percentage reduction in cell numbers of the CelA deletion strain, JWCB029, compared to the background strain JWCB018.

Mentions: To examine the phenotype of the celA deletion mutant, growth was first measured on the soluble substrate, cellobiose. Growth of the wild-type, parent, JWCB018 [21-23] (ΔpyrFA ΔcbeI), and JWCB005 [23] (ΔpyrFA) strains were virtually indistinguishable from the mutant, JWCB029 (ΔpyrFA ΔcbeI ΔcelA) (Figure 2A, B), suggesting that the celA mutant elicited no general growth defect. To determine growth on insoluble substrates, cells were stained with Acridine Orange and counted using fluorescence microscopy. We note that in our experience, growth on complex biomass as measured by optical density is not reliable, because it is difficult to distinguish cells from substrate particles.Figure 2


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)

Growth of the wild-type, JWCB005 (ΔpyrFA), JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains on various carbon sources. (A, B) growth of the wild-type (closed circles), JWCB005 (open squares), JWCB018 (open circles), and JWCB029 (open triangles) strains on cellobiose. (C-F) Growth of the JWCB018 (solid bars), and JWCB029 (open bars) strains on (C)Populus trichocarpa (poplar), (D)Panicum virgatum L. (switchgrass), (E)Arabidopsis thaliana, and (F) Avicel. (G) Growth as a percentage reduction in cell numbers of the CelA deletion strain, JWCB029, compared to the background strain JWCB018.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Growth of the wild-type, JWCB005 (ΔpyrFA), JWCB018 (ΔpyrFA ΔcbeI), and JWCB029 (ΔpyrFA ΔcbeI ΔcelA) strains on various carbon sources. (A, B) growth of the wild-type (closed circles), JWCB005 (open squares), JWCB018 (open circles), and JWCB029 (open triangles) strains on cellobiose. (C-F) Growth of the JWCB018 (solid bars), and JWCB029 (open bars) strains on (C)Populus trichocarpa (poplar), (D)Panicum virgatum L. (switchgrass), (E)Arabidopsis thaliana, and (F) Avicel. (G) Growth as a percentage reduction in cell numbers of the CelA deletion strain, JWCB029, compared to the background strain JWCB018.
Mentions: To examine the phenotype of the celA deletion mutant, growth was first measured on the soluble substrate, cellobiose. Growth of the wild-type, parent, JWCB018 [21-23] (ΔpyrFA ΔcbeI), and JWCB005 [23] (ΔpyrFA) strains were virtually indistinguishable from the mutant, JWCB029 (ΔpyrFA ΔcbeI ΔcelA) (Figure 2A, B), suggesting that the celA mutant elicited no general growth defect. To determine growth on insoluble substrates, cells were stained with Acridine Orange and counted using fluorescence microscopy. We note that in our experience, growth on complex biomass as measured by optical density is not reliable, because it is difficult to distinguish cells from substrate particles.Figure 2

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.


Related in: MedlinePlus