08/13/2015
Enhancing a microbe’s cellulolytic ability for biomass deconstruction
The in vitro activity of the Caldicellulosiruptor bescii secretome to digest lignocellulosic biomass was significantly increased with the addition of the E1 endoglucanase from Acidothermus cellulolyticus.
The Science
The most effective commercial enzyme cocktails currently used to deconstruct biomass in vitro are derived from fungal cellulase components. These fungal cellulases consist of cellobiohydrolases, endoglucanases, and β-D-glucosidases that act synergistically to release sugars from biomass for microbial conversion to products. However, these fungal cellulase components contribute significantly to overall deconstruction costs. As a potentially cost-effective alternative, C. bescii, a cellulolytic thermophile, is a prime candidate for effective consolidated bioprocessing as it contains more than 50 glycoside hydrolases including CelA, a multidomain enzyme. C. bescii’s ability to solubilize lignocellulose could be enhanced with engineering to include an endonuclease with additional activities such as E1 fromA. cellulolyticus, another cellulolytic thermophile.
The Impact
This work provides an understanding of the action and limitations of the CelA enzyme and demonstrates that CelA can act synergistically with the E1 protein to digest cellulose. These results contribute to the knowledgebase that enables enzyme engineering to generate novel enzyme mixtures for biomass deconstruction. The new information could lead to a more economical means of converting biomass to simple sugars for bioproducts production.
Summary
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 C. bescii encodes a potent set of CAZymes, found primarily as multidomain enzymes. This set of CAZymes exhibits high cellulolytic and hemicellulolytic activity on and allows 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 A. 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, U.S. Department of Energy (DOE) BioEnergy Science Center (BESC) scientists cloned and expressed the E1 gene 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 percent at 65 °C, and 12.6 percent at 75 °C. Activity against Avicel increased on average 17.5 percent at 65 °C and 16.4 percent 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 engineering additional enzymes for improved biomass deconstruction into C. bescii effectively.
Principal Investigator(s)
Janet Westpheling
University of Georgia and Oak Ridge National Laboratory
[email protected]
Funding
This research was supported as a subcontract by BESC, a DOE Bioenergy Research Center funded by the Office of Biological and Environmental Research within DOE’s Office of Science (DE-AC05-000R22725).
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.