Expression of Heterologous Endoglucanases in Caldicellulosiruptor bescii Enhances Secretome Activity

In vitro analysis of E1 and CelA shows synergistic interaction.

The Science

Currently, the most effective commercial enzyme cocktails of carbohydrate-active enzymes (CAZymes) used in vitro to pretreat biomass are derived from fungal cellulases. These cellobiohydrolases, endoglucanases, and β-d-glucosidases act synergistically to release sugars for microbial conversion. The genome of the thermophilic bacterium, Caldicellulosiruptor bescii, encodes a potent set of CAZymes, found primarily as multidomain enzymes. This set of CAZymes exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in a single protein. E1 is an endo-1,4-β-glucanase from Acidothermus cellulolyticus linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates and has been shown in vitro to work synergistically with CelA. To test if the addition of E1 to the C. bescii secretome would improve its cellulolytic activity, the E1 gene was cloned and expressed in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 % at 65 °C and 12.6 % at 75 °C. Activity against Avicel increased on average 17.5 % at 65 °C and 16.4 % at 75 °C. Thus, expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome in agreement with in vitro evidence that E1 acts synergistically with CelA to digest cellulose. This result offers the possibility of effectively engineering additional enzymes for improved biomass deconstruction into C. bescii.

Summary

The ability to deconstruct plant biomass without conventional pretreatment is an important attribute for any organism being considered for the consolidated bioprocessing of plant biomass to fuels and chemicals.  CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-D-glucosidases) that act synergistically to release sugars for microbial conversion.

Principal Investigator

Janet Westpheling
Oak Ridge National Laboratory

BER Program Manager

Shing Kwok

U.S. Department of Energy, Biological and Environmental Research (SC-33)
Biological Systems Science Division
[email protected]

References

Chung, D., J. Young, M. Cha, R. Brunecky, Y. J. Bomble, M. E. Himmel, and J. Westpheling. 2015. “Expression of the Acidothermus cellulolyticus E1 Endoglucanase in Caldicellulosiruptor bescii Enhances Its Ability to Deconstruct Crystalline Cellulose,” Biotechnology for Biofuels 8, 113. DOI: 10.1186/s13068-015-0296-x.