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United States Department of Agriculture

Agricultural Research Service

Research Project: ECOLOGY, GENOMICS, AND MANAGEMENT OF STORED PRODUCT INSECTS Title: Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae

Authors
item Shirley, Derek -
item Oppert, Cris -
item Reynolds, Todd -
item Miracle, Bethany -
item Oppert, Brenda
item Klingeman, William -
item Jurat-Fuentes, Juan Luis -

Submitted to: Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 10, 2013
Publication Date: N/A

Interpretive Summary: Some insects have enzymes that can efficiently process plant material, and thus these enzymes may have applications in the biofuels industry. One such enzyme comes from the red flour beetle, which we previously identified as a potential plant-processing enzyme in extreme conditions, such as very alkaline environments. We put the gene for this enzyme in a yeast system to further study its properties. The yeast-expressed enzyme was similar to that of the beetle enzyme, demonstrating that expression of the gene in different systems does not disrupt the activity of the protein. We speculate that this enzyme may be useful in cases where harsh alkaline conditions are necessary for processing of plant materials for applications such as biofuels.

Technical Abstract: Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the current production of fuel ethanol from lignocellulosic biomass. Cost-effectiveness of lignocellulosic ethanol production is expected to increase by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process catalyzed by yeast transformed to express efficient cellulases. While yeast represents an established heterologous expression system, insect cellulolytic enzymes have not yet been successfully expressed in yeast. To address this knowledge gap, we transformed Saccharomyces cerevisiae yeast to express the full-length cDNA encoding an endoglucanase (TcEG1) from the red flour beetle, Tribolium castaneum, and evaluated its activity. Expression of the TcEG1 cDNA in yeast was under control of the strong glyceraldehyde-3 phosphate dehydrogenase (GPD) promoter. Cultured transformed yeast secreted recombinant TcEG1 protein (rTcEG1) as a functional beta-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production, rTcEG1 activity increased at higher pH levels, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in yeast and confirm the stability of TcEG1 activity in strong alkaline environments.

Last Modified: 10/21/2014
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