Expression of a celE Gene from Clostridium thermocellum in Bacillus

The level of expression of the Clostridium thermocellum celE gene in the asporogenous Bacillus subtilis strain 1A718 did not exceed the endogenous background level. However, when transformed into sporogenous strains, celE-containing constructs allowed the cells to express a high level of thermostable carboxy-methylcellulase (CMCase) activity which was detected exclusively in the culture supernatant. The sporulation efficiency was impaired in the celE-carrying strains. Most of the thermostable CMCase activity in the recombinant strains was attributed to the stationary phase of growth, and production of the enzyme could be further enhanced by increasing the cultivation temperature from 37 degrees C to 42 degrees C. Even when expressed in an extracellular proteases deficient mutant, the protein product was cleaved in the P-T-rich Linker sequence (2 sites) and at a site downstream of the putative signal peptidase recognition site. As a consequence, the enzymatically active protein could be isolated only in a truncated form. Plasmid pHE9102, the celE-containing construct, undergoes significant structural rearrangements in Bacillus stearothermophilus strains, preventing any detectable expression.