Construction And Engineering Of Cellulases In Escherichia Coli

Cellulose, a polysaccharide of glucose units linked by β-1,4-glucosic bond, is the most abundant renewable natural biological resource on the earth. The production of biofuels and other biomaterials from cellulosic materials will bring great benefits to the sustainable development of human beings. Saccharification of cellulose usually involves pretreatment followed by enzymatic hydrolysis. The widely accepted mechanism for enzymatic hydrolysis of cellulose to glucose involves synergistic actions of three types of cellulosic enzymes:endoglucanase, exoglucanase, and β-glucosidase. Until recently, the contribution of enzyme cost to the economics of cellulosic industry continues to be a great challenge. This thesis focuses on the construction and engineering of a whole cellulase sytem in Escherichia coli, aiming at improving the catalytic efficiency of cellulosic enzymes to produce glucose from the degration of cellulose. There are six main aspects included in this research, the details are described as follows:(1) A β-glucosidase (BGL) and an endo-β-xylanase (XYN) from Trichoderma reesei, and an endoglucanase (CenA) and an exoglucanase (CbhA) from Cellulomonas fimi were cloned and expressed in E. coli. All of these four recombinant enzymes could be expressed efficently under the control of a strong T7promoter.(2) Engineering of key cellulosic enzymes de novo in E. coli provides one strategy for the rational improvement of cellulases. The bicistronic and dual-promoter constructs based on pET30a were built for the co-expression of XYN encoding gene (xyn) and BGL encoding gene (bgl) from T. reesei QM9414in E. coli. The internal ribosome binding site used in the bicistronic constructs was originally found in pET30a. In the dual-promoter constructs described here, a pET30a-derived BioBrick base vector was built for the standard assembly of two targeted genes. Compared with monocistronic constructs, the crude enzyme expressed from a bicistronic construct (xyn located upstream of bgl) and a dual-promoter construct (xyn located upstream of bgl) offered the comparable activity of two recombinant proteins. (3) A multi-promoter construct and a multi-cistronic construct harboring CbhA, CenA and BGL were built herein. Synergistic effect of three enzymes was also investigated. The results showed synergism of CenA and BGL played a critical role in degrading filter paper to glucose. Compared with monocistronic constructs, multi-promoter and multi-cistronic constructs harboring three enzymes showed significantly higher glucose yield. The specific activity of multi-promoter construct was22.5-fold and6.9-fold higher than CbhA and CenA, respectively and that of multi-cistronic construct was33.6-fold and10.3-fold higher than CbhA and CenA, respectively.(4) A novel and efficient high-throughput screening method for directed co-evolution of CenA and BGL was constructed based on the synergy of two enzymes using a bicistronic operon. To our knowledge, this is the first report for directed co-evolution of two cellulosic enzymes using an insoluble screening substrate of filter paper via their synergistic reactions. After three round mutations, six variants with increased activity were selected. A variant from the third generation offered the highest relative activity and specific activity, which was11.1-fold and2.5-fold higher than that of the wild type, respectively. Various mutations of CenA and BGL were subcloned and expressed individually. The mutations of endoglucanase and β-glucosidase offering the highest activity were combined to generate a new variant, whose relative activity and specific activity was12.3-fold and2.7-fold higher than that of the wild type, respectively.(5) A novel and efficient high-throughput screening method for directed evolution of CbhA was constructed based on the synergy of CbhA and BGL using a bicistronic operon. BGL could hydrolyze cellobiose librated by CbhA to glucose. The glucose was used as target product, and the Whatman No1filter paper was used as screening substrate in the high-throughput method. After screening4000mutants, three mutants of B1, D6and G10were found, whose specific activity was1.4-fold,1.3-fold and1.6-fold higher than that of the wild type.(6) The best positive mutants of CbhA, CenA and BGL were constructed in pET30a. The specific activity and the relative activity of constructed mutant were2.7-fold and8.2-fold higher that of the wild type, respectively. Thus, the co-engineeing of three cellulosic enzymes was achieved in this work.