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Expression of an endoglucanase from Tribolium castaneum （TcEG 1 ） in Saccharomyces cerevisiae

Insect Science 2014年第5期21卷 609-618页

作者： derek shirley Cris Oppert Todd B. Reynolds Bethany Miracle Brenda Oppert William E. Klingeman Juan Luis Jurat-Fuentes Department of Entomology and Plant Pathology University of Tennessee Department of Microbiology University of TennesseeKnoxville TN 37996 USDA-Agricultural Research Service Center for Grain and Animal Health Research Manhattan KS 66502 Department of Plant Sciences University of Tennessee Knoxville TN 37996 USA

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation （saccharification） and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum （TcEG 1）, and evaluated the activity of the transgenic product （rTcEG 1）. Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-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 with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.

关键词： biofuels insect cellulase Saccharomyces cerevisiae Tribolium castaneum yeast

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