Deletion of Caldicellulosiruptor besciiCelA reveals its crucial role in the deconstruction of lignocellulosic biomass

Abstract

BackgroundMembers 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.ResultsA 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.ConclusionsThe exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0142-6) contains supplementary material, which is available to authorized users.

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization

“…C. bescii and C. kronotskyensis have three GH48 domain-containing enzymes, while C. saccharolyticus contains only one, CelA. Deletion of CelA in C. bescii inhibited growth on cellulose and switchgrass (64). The fact that all three species contain CelA but solubilize cellulose and switchgrass to different extents implies that this cellulase may be necessary but not solely responsible for high levels of lignocellulose deconstruction.…”

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

“…Previous comparative genomics analyses confirmed the enzymatic determinants for a strongly cellulolytic phenotype, namely the presence of modular enzymes that include a GH family 48 catalytic domain and CBM family 3 (24,25). When one of these prominent modular cellulase genes (celA) was deleted from the genome, the ability of Caldicellulosiruptor bescii to grow on cellulose was significantly impacted (26). Remarkably, recent biochemical characterization of CelA indicates that the modular nature of this enzyme is beneficial such that it outperforms commercial cellulases (21).…”

Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tāpirins) from Extremely Thermophilic Caldicellulosiruptor Species

“…Unlike cellulosomal or free enzyme systems commonly found in other cellulolytic organisms, such as Clostridia (6) and Trichoderma (7), respectively, many Caldicellulosiruptor 80 carbohydrate active enzymes (CAZymes (8)) are modular and consist of combinations of catalytic and non-catalytic (e.g., carbohydrate binding module [CBM]) domains connected by proline/threonine-rich linkers in various arrangements. The most well studied example is the multifunctional cellulase, CelA, which is arranged as GH9-CBM3-CBM3-CBM3-GH48 domains, where the numbers refer to specific protein families (9)(10)(11)(12)(13)(14). The synergy between the endoglucanase 85 (GH9) and exoglucanase (GH48) domains contributes to a novel mode of action for CelA that involves physical burrowing into cellulose fibers, thereby creating cavities for further enzymatic access to the carbohydrate content of plant biomass (9).…”