【作者】 刘敏；

【导师】 于洪巍；

【作者基本信息】 浙江大学 ， 生物化工， 2013， 博士

【摘要】 有效开发利用可再生资源,是现代社会可持续发展的有力保障。生物质是最具开发潜质的一类可再生资源,其中纤维素的含量最为丰富。充分利用纤维素,将其转化为生物燃料或化学产品,对人类的可持续发展具有重要意义。要实现微生物对纤维素的有效利用,需要通过预处理和纤维素酶降解将其转变成可被微生物直接利用的可发酵性糖,主要是葡萄糖。纤维素的酶法降解是由外切酶、内切酶和p-葡萄糖苷酶协同作用来实现的。然而,纤维素酶的高成本是制约其工业化应用的重要因素。本研究旨在大肠杆菌中构建纤维素酶系共表达平台,并通过此平台对纤维素酶系进行改造,以提高纤维素酶降解纤维素产生葡萄糖的催化效率,降低其成本。为实现这一目标,本论文主要展开了以下几方面的研究工作：(1)将来源于里氏木霉的p-葡萄糖苷酶BGL和木聚糖酶XYN,来源于粪肥纤维单胞菌的内切酶CenA和外切酶CbhA,分别在大肠杆菌BL21(DE3)中实现了可溶性表达。(2)以BGL和XYN在大肠杆菌中共表达为例,改造pET30a质粒,构建双顺反子和双启动子表达盒,并研究了基因排列顺序对共表达的影响。在双顺反子表达盒中,将质粒pET30a中的核糖体结合位点及其周围的序列引入到第二段基因前面,以帮助第二段基因翻译过程的高效率进行。同时,以pET30a为基本骨架,构建了生物砖载体,利用同尾酶Xbal/PstI实现了双启动子载体的标准化组装。(3)利用改造的pET30a质粒,构建多顺反子和多启动子表达盒,实现了CbhA、 CenA和BGL在大肠杆菌中的共表达和共纯化。利用共表达的纤维素酶混合物直接降解纤维素产生葡萄糖。同时,研究了CenA与BGL之间,CbhA与BGL之间的协同作用。多启动子表达盒产葡萄糖的比活分别是CbhA和CenA的22.5倍和6.9倍。多顺反子表达盒产葡萄糖的比活分别是CbhA和CenA的33.6倍和10.3倍。(4)以CenA和BGL共表达的双顺反子载体为基础,利用两个酶之间的协同作用,以葡萄糖为目的产物,以Whatman滤纸为筛选底物,首次建立了以固体纤维素为筛选底物,可以同时筛选内切酶和p-葡萄糖苷酶的高通量筛选方法,实现了对内切酶和β-葡萄糖苷酶的共定向进化。将筛选得到的活性最高的CenA和BGL突变体通过一步亚克隆整合到一起,产生了一个新的突变体A12-H1。突变体A12-H1降解纤维素产生葡萄糖的相对活性和比活分别为野生型的12.3倍和2.7倍。(5)以CbhA和BGL共表达的双顺反子载体为基础,利用两个酶之间的协同作用,以葡萄糖为目的产物,以Whatman滤纸为筛选底物,首次建立了以固体纤维素为筛选底物,针对外切酶的高通量筛选方法,提高了其催化效率。筛选了4000个突变子后,得到了三个阳性突变体B1,D6和G10,其降解滤纸产生葡萄糖的比活分别提高了40%,32%和60%。(6)将定向改造后的CbhA突变体G10和CenA-BGL突变体A12-H1整合到质粒pET30a中,实现了对纤维素酶系的改造。整合得到的突变体降解纤维素产生葡萄糖的比活(356.9mU/mg)是野生型的2.7倍,其粗酶的相对活性(145.7mU/ml)是野生型的8.2倍。更多还原

【Abstract】 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.更多还原