Abstract
Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.
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Acknowledgments
The authors are grateful for the expert assistance of Sarah Ouanounou Moraïs throughout the stages of preparation of this manuscript. This research was supported by the Brazilian friends of the Weizmann institute of science Alternative Energy Research Initiative (research grants from Mr. Charles Rothschild, Mr. Mario Fleck, and Roberto, and Renata Ruhman), and by grants from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel and by the Israel Science Foundation (Grant nos 966/09 and 159/07). E.A.B. holds The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry.
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Caspi, J., Barak, Y., Haimovitz, R. et al. Thermobifida fusca exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A. Syst Synth Biol 4, 193–201 (2010). https://doi.org/10.1007/s11693-010-9056-1
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DOI: https://doi.org/10.1007/s11693-010-9056-1