Cloning And Coexpression Of Thermostable Endoglucanase Gene Andβ-Glucosidase Genes In Bacillus Subtilis

Bacillus subtilis, a gram-positive bacterium, is considered to be a potential strain in the biorefinery with numerous advantages, such as low-nutrient requirement, growing fast, useing hexose and pentose, efficient protein secretion ability, no endotoxin in cell wall, industrial safety, etc. The secretory coexpression of multiple cellulase genes in B.subtilis is important to reduce the cost of cellulase using in the biorefinery industrial.In this study, bglA gene from Bacillus polymyxa encoding P-glucosidases was cloned into pMA5and expressed in B.subtilis WB600and WB800. The recombinant strain can grow well in the medium that cellobiose as its only carbon source. In order to realize the secretory expression of cellulase genes, the pP43JM2shuttle vector which carrying NprB signal peptide of the B.subtilis neutral protease was used as the expression vector for expressing the bglA and bglB genes from Bacillus polymyxa encoding P-glucosidases in B.subtilis WB800, respectively. The results showed that the bglB gene was expressed and secreted into the broth. Cellulase is a kind of multiple enzymes system and requires synergistic effect among various cellulase components, so the secretory coexpression of cellulase genes in B.subtilis is beneficial to the high cellulose degradation. In order to realize the secretory coexpression of cellulase genes, the recombinant plasmids harboring the celA gene from Clostridium thermocellum encoding endoglucanase and the bglA, bglB genes encoding P-glucosidases were constructed, respectively, and then transformed into B.subtilis WB800. The results showed that the coexpression of these enzymes was viable in B.subtilis WB800. The endoglucanase and the p-glucosidases encoded by bglB were secreted into the culture broth successfully, while the β-glucosidases encoding by bglA was not and a high intracellular cellobiase activity of bglA was detected. Efficient coexpression of cellulase enzymes into B.subtilis paved the way of constructing consolidated bioprocessing strains for bulk chemicals or bioenergy production economically.