Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli
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Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.
KeywordsFungal cellulase Fusion protein Heterologous protein expression Membrane localization
This study was supported by The National Science Foundation (NSF) CAREER Program (CBET-0952875) and funding from the Princeton University. The authors would like to thank Professor Natividad Ruiz (now at Ohio State University) and Dr. Juliana Malinverni from the Silhavy Lab at the Princeton University for providing antibodies and their expertise on sucrose gradient centrifugation. We would also like to thank the Shenk Lab at Princeton University for their help with ultracentrifugation.
- Denman S, Xue GP, Patel B (1996) Characterization of a Neocallimastix patriciarum cellulase cDNA (celA) homologous to Trichoderma reesei cellobiohydrolase II. Appl Environ Microb 62:1889–1896Google Scholar
- Xu Z, Lee SY (1999) Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Appl Environ Microb 65:5142–5147Google Scholar