Secretive Expression Of Cellulase Enzyme And Its Application In Biorefinery Processes

Consolidated bioprocessing process (CBP) is an important strategy in biorefinery processes using lignocellulose as the substrate. CBP microbes must own the ability of cellulase production, cellulose hydrolysis, and fermentation to reduce the cost of biorefinery process through the decrease of the cellulase dosage. Few wild type strains are capable of CBP functions with all the three properties, therefore metabolic engineering is the major focus for developing CBP strains, and great efforts have been invested into the this field.Escherichia coli is not capable of cellulose degradation and ethanol fermentation, but it could be genetically engineered to behanve the CBP properties. In this thesis, E. coli was engineered by integrating two genes from Zymomonas mobilis, pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase gene (adhB), into the genome of E. coli JM109 to obtain the ethanologenic E. coli recombinant. Then, the β-glucosidase gene bglB from Bacillus polymyxa, as well as the endoglucanase gene celA and the exoglucanase gene cbhA from Clostridium thermocellum, were heterologously expressed in the recombinant E. coli by using the screened promoters and signal pepteids. The recombinant E. coli strains containing the genes demonstrated the corresponding intracellular cellulase activity and CBP fermentability. Moreover, the surfactants were used to enhance the release of cellulase proteins from the periplasm space into the medium by increasing the permeability of the cell membrane and promoting the secretion of the cellulase proteins. The addition of 0.5% EDTA showed the most obviously improvement in the ethanol fermentation by using cellobiose as the substrate.Zymomonas mobilis is a gram-negative facultative bacterium for production of ethanol at a high yield through ED pathway using glucose, fructose and sucrose as the substrate, as well as lower biomass production, facultative oxygen requirement and the tolerance to the toxicity of the final product. However, Z.mobilis is unable to degrade the lignocellulose. In this thesis, new expression strategies were investigated and the β-glucosidase gene bglB from Bacillus polymyxa was secretively expressed in Z. mobilis. The endogenous cellulase gene ZMO1086 was over-expressed in Z. mobilis to test the possibility and secretion situation of cellulase. Then the signal peptide of ZMO1086 was used to facilitate the secretive expression of bglB in Z. mobilis. We also found that the Bg1B protein could be secreted through the form of fusion protein by fused with the levansucrase sacB gene of Z. mobilis. The recombinant Z. mobilis expressing the bglB gene showed the ability of cellobiose utilization. The study of the new expression strategies and the Bg1B secretion in Z. mobilis improved the understanding on the cellulase expression and provided some new methods for the future secretive expression of cellulase in Z. mobilis.Bacillus subtilis is frequently used for the secretive expression of various heterogeneous proteins for its excellent protein secretive capacity. It also performance well in the fermantaion process to produce lactic acid or butane for its outstanding utilization of hexose and pentose, fast growth rate, and low nutrient need. A β-1,3-1,4-glucanase gene licB was obtained from Clostridium thermocellum and secretively expressed in Bacillus subtilis. LicB showed the optimal β-1,3-1,4-glucanase activity at 80℃ and pH at 7 in a wide pH range from 4 to 11. The secreted protein retained more than 50% activity when being incubated at 70℃ for 5 hours. It is active in the condition with different metal ion except for Cu2+ and Fe2+ The thermostability and pH adaptability make LicB can be widely used in the industrial processing, and the excellent expression and secretion of cellulase in Bacillus subtilis may play an important role in the CBP research.Conclusively, the genetic engineering research on the potential CBP strains of Zymomonas mobilis, E. coli and Bacillus subtilis were investigated. The secretion of cellulase through expressing different cellulase gene in different strains realized the CBP fermentation capacity at different extent. The study paved the way of constructing and applying CBP strain in biorefinery processes.