Leakage of Cellulomonas fimi cellulases from Escherichia coli

Transcription of the cenA gene of Cellulomonas fimi, encoding endoglucanase A (EngA), from the lac promoter of pUC18 rather than from the tet promoter of pBR322 increased the level of expression of the gene in Escherichia coli some 800-fold. Most of the EngA activity was exported to the periplasm, even though the EngA leader peptide in the construct was 9 amino acids longer than usual. The increased level of expression of cenA resulted in the temperature-dependent leakage of periplasmic proteins, including EngA and β-lactamase, into the culture medium. In contrast to C. fimi, which processes pro-EngA at a single site, E. coli processed it at two distinct sites 3 amino acids apart. Increasing the level of expression of cex, encoding an exoglucanase (Exg) of C. fimi, to a comparable extent also led to the temperature-dependent leakage of Exg into the culture medium.

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