Screening For Cellobiohydrolase Genes And The Secretion Of Thermostable Cellulase In Bacillus Subtilis

The screening for the powerful cellulase components remains to be a challenging work for the biorefinery research and development. The engineered bacterial strains for production of cellulase would provide many advantages, such as the low nutrients requirement, easy manipulation of genetic engineering, etc.In this study, about 20 cellobiohydrolase genes sourced from Clostridium thermocellum DSM 1237, Clostridium cellulolyticum DSM 5812 and Xanthomonas campestris pv. campestris strain 8004, were tested to be expressed in Escherichia coli BL21 (DE3)/pET-28a(+) system for activity evaluation.In order to realize the secretion of cellulases extracellularly, Bacillus subtilis WB600 (WB800)/pP43JM2 expression system was constructed. When the targeting genes including cellobiohydrolase genes from Clostridium thermocellum, endoglucanse genes from Clostridium thermocellum, Erwinia carotovora subsp. and Thermomonospora fusca.were cloned into pP43JM2 and expressed in B. subtilis WB600 and WB800, all the cellulases, except for celO, were expressed and secreted into the broth and showed the activities with phosphoric acid swollen cellulose as the substrate. For cellobiohydrolases, the enzyme activities from WB800 were much higher than that from WB600, CbhA can produce 300 mg/L reducing sugars. While for endoglucanase CelA, the reducing sugars reached 1250 mg/L. Through the combination of CbhA and CelA, it showed a clear synergistic effect towards Avicel and dilute-acid pretreated corn stover.The established B. subtilis expression system was proven to be a useful platform for secretive expression and would be applied for multiple cellulase production in the future research. Efficient secretion of active cellulase is the first step in the development of B. subtilis consolidated bioprocessing cells that can produce cellulase, hydrolyze cellulose, and ferment the soluble sugars to products.